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Sample records for precursor cells derived

  1. Enrichment of skin-derived neural precursor cells from dermal cell populations by altering culture conditions.

    PubMed

    Bayati, Vahid; Gazor, Rohoullah; Nejatbakhsh, Reza; Negad Dehbashi, Fereshteh

    2016-01-01

    As stem cells play a critical role in tissue repair, their manipulation for being applied in regenerative medicine is of great importance. Skin-derived precursors (SKPs) may be good candidates for use in cell-based therapy as the only neural stem cells which can be isolated from an accessible tissue, skin. Herein, we presented a simple protocol to enrich neural SKPs by monolayer adherent cultivation to prove the efficacy of this method. To enrich neural SKPs from dermal cell populations, we have found that a monolayer adherent cultivation helps to increase the numbers of neural precursor cells. Indeed, we have cultured dermal cells as monolayer under serum-supplemented (control) and serum-supplemented culture, followed by serum free cultivation (test) and compared. Finally, protein markers of SKPs were assessed and compared in both experimental groups and differentiation potential was evaluated in enriched culture. The cells of enriched culture concurrently expressed fibronectin, vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors as compared to control culture. In addition, they possessed a multipotential capacity to differentiate into neurogenic, glial, adipogenic, osteogenic and skeletal myogenic cell lineages. It was concluded that serum-free adherent culture reinforced by growth factors have been shown to be effective on proliferation of skin-derived neural precursor cells (skin-NPCs) and drive their selective and rapid expansion.

  2. Human iPSC Derived GABA Ergic Precursor Cell Therapy for Chronic Epilepsy

    DTIC Science & Technology

    2016-10-01

    AWARD NUMBER: W81XWH-14-1-0558 TITLE: Human iPSC-Derived GABA-Ergic Precursor Cell Therapy for Chronic Epilepsy PRINCIPAL INVESTIGATOR: Ashok K...SUBTITLE Human iPSC-Derived GABA-Ergic Precursor Cell Therapy for Chronic Epilepsy 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-14-1-0558 5c...medial ganglionic eminence (hMGE)-like precursor cells generated from the human induced pluripotent stem cells (hiPSCs) into the hippocampus of

  3. Human iPSC-Derived GABA Ergic Precursor Cell Therapy for Chronic Epilepsy

    DTIC Science & Technology

    2015-10-01

    AWARD NUMBER: W81XWH-14-1-0558 TITLE: Human iPSC-Derived GABA-Ergic Precursor Cell Therapy for Chronic Epilepsy PRINCIPAL INVESTIGATOR: Ashok K...AND SUBTITLE 5a. CONTRACT NUMBER Human iPSC-Derived GABA-Ergic Precursor Cell Therapy for Chronic Epilepsy 5b. GRANT NUMBER W81XWH-14-1-0558 5c...exhibiting chronic temporal lobe epilepsy (TLE) would: (1) greatly diminish the frequency and intensity of spontaneous recurrent seizures (SRS, Specific

  4. Derivation of Neural Precursor Cells from Human Embryonic Stem Cells for DNA Methylomic Analysis.

    PubMed

    Roubal, Ivan; Park, Sun Joo; Kim, Yong

    2016-01-01

    Embryonic stem cells are self-renewing pluripotent cells with competency to differentiate into all three-germ lineages. Many studies have demonstrated the importance of genetic and epigenetic molecular mechanisms in the maintenance of self-renewal and pluripotency. Stem cells are under unique molecular and cellular regulations different from somatic cells. Proper regulation should be ensured to maintain their unique self-renewal and undifferentiated characteristics. Understanding key mechanisms in stem cell biology will be important for the successful application of stem cells for regenerative therapeutic medicine. More importantly practical use of stem cells will require our knowledge on how to properly direct and differentiate stem cells into the necessary type of cells. Embryonic stem cells and adult stem cells have been used as study models to unveil molecular and cellular mechanisms in various signaling pathways. They are especially beneficial to developmental studies where in vivo molecular/cellular study models are not available. We have derived neural stem cells from human embryonic stem cells as a model to study the effect of teratogen in neural development. We have tested commercial neural differentiation system and successfully derived neural precursor cells exhibiting key molecular features of neural stem cells, which will be useful for experimental application.

  5. Induction of Skin-Derived Precursor Cells from Human Induced Pluripotent Stem Cells.

    PubMed

    Sugiyama-Nakagiri, Yoriko; Fujimura, Tsutomu; Moriwaki, Shigeru

    2016-01-01

    The generation of full thickness human skin from dissociated cells is an attractive approach not only for treating skin diseases, but also for treating many systemic disorders. However, it is currently not possible to obtain an unlimited number of skin dermal cells. The goal of this study was to develop a procedure to produce skin dermal stem cells from induced pluripotent stem cells (iPSCs). Skin-derived precursor cells (SKPs) were isolated as adult dermal precursors that could differentiate into both neural and mesodermal progenies and could reconstitute the dermis. Thus, we attempted to generate SKPs from iPSCs that could reconstitute the skin dermis. Human iPSCs were initially cultured with recombinant noggin and SB431542, an inhibitor of activin/nodal and TGFβ signaling, to induce neural crest progenitor cells. Those cells were then treated with SKP medium that included CHIR99021, a WNT signal activator. The induction efficacy from neural crest progenitor cells to SKPs was more than 97%. No other modifiers tested were able to induce those cells. Those human iPSC-derived SKPs (hiPSC-SKPs) showed a similar gene expression signature to SKPs isolated from human skin dermis. Human iPSC-SKPs differentiated into neural and mesodermal progenies, including adipocytes, skeletogenic cell types and Schwann cells. Moreover, they could be induced to follicular type keratinization when co-cultured with human epidermal keratinocytes. We here provide a new efficient protocol to create human skin dermal stem cells from hiPSCs that could contribute to the treatment of various skin disorders.

  6. Skin-derived precursor cells promote wound healing in diabetic mice.

    PubMed

    Sato, Hideyoshi; Ebisawa, Katsumi; Takanari, Keisuke; Yagi, Shunjiro; Toriyama, Kazuhiro; Yamawaki-Ogata, Aika; Kamei, Yuzuru

    2015-01-01

    Impaired wound healing as one of the complications arising from diabetes mellitus is a serious clinical issue. Recently, various cell therapies have been reported for promotion of wound healing. Skin-derived precursor cells (SKPs) are multipotent adult stem cells with the tendency to differentiate into neurons. We investigated the potency of promoting diabetic wound healing by the application of SKPs. Skin-derived precursor cells isolated from diabetic murine skin were cultured in sphere formation medium. At passage 2, they were suspended in phosphate-buffered saline (PBS), and applied topically to full-thickness excisional cutaneous wounds in diabetic mice. Application of PBS served as controls (n = 21 for each group; n = 42 total). Time to closure and percentage closure were calculated by morphometry. Wounds were harvested at 10 and 28 days and then processed, sectioned, and stained (CD31, α-smooth muscle actin, and neurofilament heavy chain) to quantify vascularity and neurofilaments. Wounds treated with SKPs demonstrated a significantly decreased time to closure (18.63 days) compared with PBS-control wounds (21.72 days, P < 0.01), and a significant improvement in percentage closure at 7, 10, 14, and 18 days compared with PBS-control wounds (P < 0.01). Histological analysis showed that the Capillary Score (the number of vessels/mm2) was significantly higher in SKP-treated wounds at day 10 but not at day 28. Nerve Density (the number of neurofilaments/mm2) had increased significantly in SKP-treated wounds at day 28 compared with control group. Some applied SKPs were stained by neurofilament heavy chain, which demonstrates that SKPs directly differentiated into neurons. Skin-derived precursor cells promoted diabetic wound healings through vasculogenesis at the early stage of wound healing. Skin-derived precursor cells are a possible therapeutic tool for diabetic impaired wound healing.

  7. Alcohol-Induced Molecular Dysregulation in Human Embryonic Stem Cell-Derived Neural Precursor Cells.

    PubMed

    Kim, Yi Young; Roubal, Ivan; Lee, Youn Soo; Kim, Jin Seok; Hoang, Michael; Mathiyakom, Nathan; Kim, Yong

    Adverse effect of alcohol on neural function has been well documented. Especially, the teratogenic effect of alcohol on neurodevelopment during embryogenesis has been demonstrated in various models, which could be a pathologic basis for fetal alcohol spectrum disorders (FASDs). While the developmental defects from alcohol abuse during gestation have been described, the specific mechanisms by which alcohol mediates these injuries have yet to be determined. Recent studies have shown that alcohol has significant effect on molecular and cellular regulatory mechanisms in embryonic stem cell (ESC) differentiation including genes involved in neural development. To test our hypothesis that alcohol induces molecular alterations during neural differentiation we have derived neural precursor cells from pluripotent human ESCs in the presence or absence of ethanol treatment. Genome-wide transcriptomic profiling identified molecular alterations induced by ethanol exposure during neural differentiation of hESCs into neural rosettes and neural precursor cell populations. The Database for Annotation, Visualization and Integrated Discovery (DAVID) functional analysis on significantly altered genes showed potential ethanol's effect on JAK-STAT signaling pathway, neuroactive ligand-receptor interaction, Toll-like receptor (TLR) signaling pathway, cytokine-cytokine receptor interaction and regulation of autophagy. We have further quantitatively verified ethanol-induced alterations of selected candidate genes. Among verified genes we further examined the expression of P2RX3, which is associated with nociception, a peripheral pain response. We found ethanol significantly reduced the level of P2RX3 in undifferentiated hESCs, but induced the level of P2RX3 mRNA and protein in hESC-derived NPCs. Our result suggests ethanol-induced dysregulation of P2RX3 along with alterations in molecules involved in neural activity such as neuroactive ligand-receptor interaction may be a molecular event

  8. Alcohol-Induced Molecular Dysregulation in Human Embryonic Stem Cell-Derived Neural Precursor Cells

    PubMed Central

    Kim, Yi Young; Roubal, Ivan; Lee, Youn Soo; Kim, Jin Seok; Hoang, Michael; Mathiyakom, Nathan; Kim, Yong

    2016-01-01

    Adverse effect of alcohol on neural function has been well documented. Especially, the teratogenic effect of alcohol on neurodevelopment during embryogenesis has been demonstrated in various models, which could be a pathologic basis for fetal alcohol spectrum disorders (FASDs). While the developmental defects from alcohol abuse during gestation have been described, the specific mechanisms by which alcohol mediates these injuries have yet to be determined. Recent studies have shown that alcohol has significant effect on molecular and cellular regulatory mechanisms in embryonic stem cell (ESC) differentiation including genes involved in neural development. To test our hypothesis that alcohol induces molecular alterations during neural differentiation we have derived neural precursor cells from pluripotent human ESCs in the presence or absence of ethanol treatment. Genome-wide transcriptomic profiling identified molecular alterations induced by ethanol exposure during neural differentiation of hESCs into neural rosettes and neural precursor cell populations. The Database for Annotation, Visualization and Integrated Discovery (DAVID) functional analysis on significantly altered genes showed potential ethanol’s effect on JAK-STAT signaling pathway, neuroactive ligand-receptor interaction, Toll-like receptor (TLR) signaling pathway, cytokine-cytokine receptor interaction and regulation of autophagy. We have further quantitatively verified ethanol-induced alterations of selected candidate genes. Among verified genes we further examined the expression of P2RX3, which is associated with nociception, a peripheral pain response. We found ethanol significantly reduced the level of P2RX3 in undifferentiated hESCs, but induced the level of P2RX3 mRNA and protein in hESC-derived NPCs. Our result suggests ethanol-induced dysregulation of P2RX3 along with alterations in molecules involved in neural activity such as neuroactive ligand-receptor interaction may be a molecular event

  9. Morphological and electrophysiological features of mature neurons in differentiated skin-derived precursor cells.

    PubMed

    Liebmann, L; Beetz, C; Thorwarth, M; Deufel, T; Hübner, Ca

    2012-01-01

    In vitro modelling of neuronal pathologies is, in particular, demanding and a lot of efforts have been undertaken to differentiate skin derived precursor cells into neuronal cells. However, so far all attempts did not result in cells with functional features of neurons like the ability to generate action potentials or synaptic activity. Here, we report that simple modifications of the protocols result in neuronal cells that display action potentials and synaptic activity. We think that our observation is an important step to model individual neuronal pathologies in vitro.

  10. Functional signature of human islet-derived precursor cells compared to bone marrow-derived mesenchymal stem cells.

    PubMed

    Limbert, Catarina; Ebert, Regina; Schilling, Tatjana; Path, Gunter; Benisch, Peggy; Klein-Hitpass, Ludger; Seufert, Jochen; Jakob, Franz

    2010-05-01

    Pancreatic islet beta-cell replenishment can be driven by epithelial cells from exocrine pancreas via epithelial-mesenchymal transition (EMT) and the reverse process MET, while specified pancreatic mesenchymal cells control islet cell development and maintenance. The role of human islet-derived precursor cells (hIPCs) in regeneration and support of endocrine islets is under investigation. Here, we analyzed hIPCs as to their immunophenotype, multilineage differentiation capacity, and gene profiling, in comparison to human bone marrow-derived mesenchymal stem cells (hBM-MSCs). hIPCs and hBM-MSCs display a common mesenchymal character and express lineage-specific marker genes upon induction toward pancreatic endocrine and mesenchymal pathways of differentiation. hIPCs can go further along endocrine pathways while lacking some core mesenchymal differentiation attributes. Significance analysis of microarray (SAM) from 5 hBM-MSC and 3 hIPC donors mirrored such differences. Candidate gene cluster analysis disclosed differential expression of key lineage regulators, indicated a HoxA gene-associated positional memory in hIPCs and hBM-MSCs, and showed as well a clear transition state from mesenchyme to epithelium or vice versa in hIPCs. Our findings raise new research platforms to further clarify the potential of hIPCs to undergo complete MET thus contributing to islet cell replenishment, maintenance, and function.

  11. An Engineered Cardiac Reporter Cell Line Identifies Human Embryonic Stem Cell-Derived Myocardial Precursors

    PubMed Central

    Mihardja, Shirley S.; Liszewski, Walter; Erle, David J.; Lee, Randall J.; Bernstein, Harold S.

    2011-01-01

    Unlike some organs, the heart is unable to repair itself after injury. Human embryonic stem cells (hESCs) grow and divide indefinitely while maintaining the potential to develop into many tissues of the body. As such, they provide an unprecedented opportunity to treat human diseases characterized by tissue loss. We have identified early myocardial precursors derived from hESCs (hMPs) using an α-myosin heavy chain (αMHC)-GFP reporter line. We have demonstrated by immunocytochemistry and quantitative real-time PCR (qPCR) that reporter activation is restricted to hESC-derived cardiomyocytes (CMs) differentiated in vitro, and that hMPs give rise exclusively to muscle in an in vivo teratoma formation assay. We also demonstrate that the reporter does not interfere with hESC genomic stability. Importantly, we show that hMPs give rise to atrial, ventricular and specialized conduction CM subtypes by qPCR and microelectrode array analysis. Expression profiling of hMPs over the course of differentiation implicate Wnt and transforming growth factor-β signaling pathways in CM development. The identification of hMPs using this αMHC-GFP reporter line will provide important insight into the pathways regulating human myocardial development, and may provide a novel therapeutic reagent for the treatment of cardiac disease. PMID:21245908

  12. Isolation of Mature (Peritoneum-Derived) Mast Cells and Immature (Bone Marrow-Derived) Mast Cell Precursors from Mice

    PubMed Central

    Meurer, Steffen K.; Neß, Melanie; Weiskirchen, Sabine; Kim, Philipp; Tag, Carmen G.; Kauffmann, Marlies; Huber, Michael; Weiskirchen, Ralf

    2016-01-01

    Mast cells (MCs) are a versatile cell type playing key roles in tissue morphogenesis and host defence against bacteria and parasites. Furthermore, they can enhance immunological danger signals and are implicated in inflammatory disorders like fibrosis. This granulated cell type originates from the myeloid lineage and has similarities to basophilic granulocytes, both containing large quantities of histamine and heparin. Immature murine mast cells mature in their destination tissue and adopt either the connective tissue (CTMC) or mucosal (MMC) type. Some effector functions are executed by activation/degranulation of MCs which lead to secretion of a typical set of MC proteases (MCPT) and of the preformed or newly synthesized mediators from its granules into the local microenvironment. Due to the potential accumulation of mutations in key signalling pathway components of corresponding MC cell-lines, primary cultured MCs are an attractive mean to study general features of MC biology and aspects of MC functions relevant to human disease. Here, we describe a simple protocol for the simultaneous isolation of mature CTMC-like murine MCs from the peritoneum (PMCs) and immature MC precursors from the bone marrow (BM). The latter are differentiated in vitro to yield BM-derived MCs (BMMC). These cells display the typical morphological and phenotypic features of MCs, express the typical MC surface markers, and can be propagated and kept in culture for several weeks. The provided protocol allows simple amplification of large quantities of homogenous, non-transformed MCs from the peritoneum and bone marrow-derived mast cells for cell- and tissue-based biomedical research. PMID:27337047

  13. Learning-induced synaptic potentiation in implanted neural precursor cell-derived neurons

    PubMed Central

    Park, Kyungjoon; Heo, Hwon; Han, Ma Eum; Choi, Kyuhyun; Yi, Jee Hyun; Kang, Shin Jung; Kwon, Yunhee Kim; Shin, Ki Soon

    2015-01-01

    Neuronal loss caused by neurodegenerative diseases, traumatic brain injury and stroke results in cognitive dysfunctioning. Implantation of neural stem/precursor cells (NPCs) can improve the brain function by replacing lost neurons. Proper synaptic integration following neuronal differentiation of implanted cells is believed to be a prerequisite for the functional recovery. In the present study, we characterized the functional properties of immortalized neural progenitor HiB5 cells implanted into the rat hippocampus with chemically induced lesion. The implanted HiB5 cells migrated toward CA1 pyramidal layer and differentiated into vGluT1-positive glutamatergic neurons with morphological and electrophysiological properties of endogenous CA1 pyramidal cells. Functional synaptic integration of HiB5 cell-derived neurons was also evidenced by immunohistochemical and electrophysiological data. Lesion-caused memory deficit was significantly recovered after the implantation when assessed by inhibitory avoidance (IA) learning. Remarkably, IA learning preferentially produced long-term potentiation (LTP) at the synapses onto HiB5 cell-derived neurons, which occluded paring protocol-induced LTP ex vivo. We conclude that the implanted HiB5 cell-derived neurons actively participate in learning process through LTP formation, thereby counteracting lesion-mediated memory impairment. PMID:26634434

  14. Hepatic precursors derived from murine embryonic stem cells contribute to regeneration of injured liver.

    PubMed

    Heo, Jeonghoon; Factor, Valentina M; Uren, Tania; Takahama, Yasushi; Lee, Ju-Seog; Major, Marian; Feinstone, Stephen M; Thorgeirsson, Snorri S

    2006-12-01

    We established an efficient system for differentiation, expansion and isolation of hepatic progenitor cells from mouse embryonic stem (ES) cells and evaluated their capacity to repopulate injured liver. Using mouse ES cells transfected with the green fluorescent protein (GFP) reporter gene regulated by albumin (ALB) enhancer/promoter, we found that a serum-free chemically defined medium supports formation of embryoid bodies (EBs) and differentiation of hepatic lineage cells in the absence of exogenous growth factors or feeder cell layers. The first GFP+ cells expressing ALB were detected in close proximity to "beating" myocytes after 7 days of EB cultures. GFP+ cells increased in number, acquired hepatocyte-like morphology and hepatocyte-specific markers (i.e., ALB, AAT, TO, and G6P), and by 28 days represented more than 30% of cells isolated from EB outgrowths. The FACS-purified GFP+ cells developed into functional hepatocytes without evidence of cell fusion and participated in the repairing of diseased liver when transplanted into MUP-uPA/SCID mice. The ES cell-derived hepatocytes were responsive to normal growth regulation and proliferated at the same rate as the host hepatocytes after an additional growth stimulus from CCl(4)-induced liver injury. The transplanted GFP+ cells also differentiated into biliary epithelial cells. In conclusion, a highly enriched population of committed hepatocyte precursors can be generated from ES cells in vitro for effective cell replacement therapy.

  15. Efficient delivery of human single fiber-derived muscle precursor cells via biocompatible scaffold.

    PubMed

    Boldrin, Luisa; Malerba, Alberto; Vitiello, Libero; Cimetta, Elisa; Piccoli, Martina; Messina, Chiara; Gamba, Pier Giorgio; Elvassore, Nicola; De Coppi, Paolo

    2008-01-01

    The success of cell therapy for skeletal muscle disorders depends upon two main factors: the cell source and the method of delivery. In this work we have explored the therapeutic potential of human muscle precursor cells (hMPCs), obtained from single human muscle fibers, implanted in vivo via micropatterned scaffolds. hMPCs were initially expanded and characterized in vitro by immunostaining and flow cytometric analysis. For in vivo studies, hMPCs were seeded onto micropatterned poly-lactic-glycolic acid 3D-scaffolds fabricated using soft-lithography and thermal membrane lamination. Seeded scaffolds were then implanted in predamaged tibialis anterior muscles of CD1 nude mice; hMPCs were also directly injected in contralateral limbs as controls. Similarly to what we previously described with mouse precursors cells, we found that hMPCs were able to participate in muscle regeneration and scaffold-implanted muscles contained a greater number of human nuclei, as revealed by immunostaining and Western blot analyses. These results indicate that hMPCs derived from single fibers could be a good and reliable cell source for the design of therapeutic protocols and that implantation of cellularized scaffolds is superior to direct injection for the delivery of myogenic cells into regenerating skeletal muscle.

  16. Transplanted skin-derived precursor stem cells generate enteric ganglion-like structures in vivo.

    PubMed

    Wagner, Justin P; Sullins, Veronica F; Dunn, James C Y

    2014-08-01

    Hirschsprung's disease is characterized by a developmental arrest of neural crest cell migration, causing distal aganglionosis. Transplanted cells derived from the neural crest may regenerate enteric ganglia in this condition. We investigated the potential of skin-derived precursor cells (SKPs) to engraft and to differentiate into enteric ganglia in aganglionic rat intestine in vivo. Adult Lewis rat jejunal segments were separated from intestinal continuity and treated with benzalkonium chloride to induce aganglionosis. Ganglia were identified via immunohistochemical stains for S100 and β-III tubulin (TUJ1). SKPs were procured from neonatal Lewis rats expressing enhanced green fluorescent protein (GFP) and cultured in neuroglial-selective media. SKP cell line expansion was quantified, and immunophenotypes were assessed by immunocytochemistry. Aganglionic segments underwent SKP transplantation 21-79days after benzalkonium chloride treatment. The presence of GFP+cells, mature neurons, and mature glia was evaluated at posttransplant days 1, 6, and 9. Benzalkonium chloride-induced aganglionosis persisted for at least 85days. Prior to differentiation, SKPs expressed S100, denoting neural crest lineage, and nestin, a marker of neuronal precursors. Differentiated SKPs in vitro expressed GFAP, a marker of glial differentiation, as well as TUJ1 and several enteric neurotransmitters. After transplantation, GFP+structures resembling ganglia were identified between longitudinal and circular smooth muscle layers. SKPs are capable of engraftment, migration, and differentiation within aganglionic rodent intestine in vivo. Differentiated SKPs generate structures that resemble enteric ganglia. Our observations suggest that SKPs represent a potential gangliogenic therapeutic agent for Hirschsprung's disease. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Measurement of precursor miRNA in exosomes from human ESC-derived mesenchymal stem cells.

    PubMed

    Chen, Tian Sheng; Lim, Sai Kiang

    2013-01-01

    Mesenchymal stem cells (MSCs) derived from human embryonic stem cells (ESCs) have been shown to secrete exosomes that are cardioprotective against myocardial ischemia reperfusion injury in a mouse model. To elucidate this cardioprotective mechanism, we have characterized the protein, nucleic acid, and lipid composition of MSC exosomes. Here we describe the isolation and analysis of RNA in MSC exosome. We have previously reported that RNAs in MSC exosome are primarily small RNA molecules of <300 nt and they include many miRNAs. Many of these miRNAs are in the precursor form suggesting that pre-miRNAs, and not mature miRNAs are preferentially loaded into exosomes. The protocols described here include assays to ascertain the presence of pre-miRNAs, profiling of miRNA and pre-miRNA, and quantitative estimation of mature and pre-miRNA.

  18. HIV impairs CD34+-derived monocytic precursor differentiation into functional dendritic cells.

    PubMed

    Bordoni, V; Castelli, G; Montesoro, E; Federico, M; Sacchi, A; Morsilli, O; Agrati, C; Martini, F; Chelucci, C

    2013-01-01

    Dendritic cells (DCs) perform a basic role in the immune system by allowing the initiation of the primary T-cell-dependent immune response. Given previous indirect evidence that DC maturation and function are impaired by HIV, we have developed an in vitro culture system in order to verify the effect of HIV infection on DC function during the development from hematopoietic progenitors. Considering that monocytic (Mo) differentiating cells efficiently replicate monocytotropic HIV, we examined whether HIV-infected monocytic precursors (MoP) were able to generate functional DCs. CD34+ hematopoietic progenitor cells (HPCs) were induced along Mo differentiative pathway in liquid cultures and at an early stage of culture, MoP were infected with M-tropic BaL HIV strain, and after 2 days they were switched to DC differentiation with GM-CSF and IL-4. Derived DCs were actively infected, as detected by HIV-p24 production. HIV did not significantly affect cell viability, but induced a reduction in cell proliferation and an inefficient functional activity in terms of uptake capability and stimulation of allogenic T cells. These results indicate that HIV-infected MoP lost the capacity to generate functional DCs, and this may represent one of the many mechanisms of immunosuppression exploited by HIV.

  19. iPSC-derived neural precursor cells: potential for cell transplantation therapy in spinal cord injury.

    PubMed

    Nagoshi, Narihito; Okano, Hideyuki

    2017-10-09

    A number of studies have demonstrated that transplantation of neural precursor cells (NPCs) promotes functional recovery after spinal cord injury (SCI). However, the NPCs had been mostly harvested from embryonic stem cells or fetal tissue, raising the ethical concern. Yamanaka and his colleagues established induced pluripotent stem cells (iPSCs) which could be generated from somatic cells, and this innovative development has made rapid progression in the field of SCI regeneration. We and other groups succeeded in producing NPCs from iPSCs, and demonstrated beneficial effects after transplantation for animal models of SCI. In particular, efficacy of human iPSC-NPCs in non-human primate SCI models fostered momentum of clinical application for SCI patients. At the same time, however, artificial induction methods in iPSC technology created alternative issues including genetic and epigenetic abnormalities, and tumorigenicity after transplantation. To overcome these problems, it is critically important to select origins of somatic cells, use integration-free system during transfection of reprogramming factors, and thoroughly investigate the characteristics of iPSC-NPCs with respect to quality management. Moreover, since most of the previous studies have focused on subacute phase of SCI, establishment of effective NPC transplantation should be evaluated for chronic phase hereafter. Our group is currently preparing clinical-grade human iPSC-NPCs, and will move forward toward clinical study for subacute SCI patients soon in the near future.

  20. GDNF facilitates differentiation of the adult dentate gyrus-derived neural precursor cells into astrocytes via STAT3

    SciTech Connect

    Boku, Shuken; Nakagawa, Shin; Takamura, Naoki; Kato, Akiko; Takebayashi, Minoru; Hisaoka-Nakashima, Kazue; Omiya, Yuki; Inoue, Takeshi; Kusumi, Ichiro

    2013-05-17

    Highlights: •GDNF has no effect on ADP proliferation and apoptosis. •GDNF increases ADP differentiation into astrocyte. •A specific inhibitor of STAT3 decreases the astrogliogenic effect of GDNF. •STAT3 knockdown by lentiviral shRNA vector also decreases the astrogliogenic effect of GDNF. •GDNF increases the phosphorylation of STAT3. -- Abstract: While the pro-neurogenic actions of antidepressants in the adult hippocampal dentate gyrus (DG) are thought to be one of the mechanisms through which antidepressants exert their therapeutic actions, antidepressants do not increase proliferation of neural precursor cells derived from the adult DG. Because previous studies showed that antidepressants increase the expression and secretion of glial cell line-derived neurotrophic factor (GDNF) in C6 glioma cells derived from rat astrocytes and GDNF increases neurogenesis in adult DG in vivo, we investigated the effects of GDNF on the proliferation, differentiation and apoptosis of cultured neural precursor cells derived from the adult DG. Data showed that GDNF facilitated the differentiation of neural precursor cells into astrocytes but had no effect on their proliferation or apoptosis. Moreover, GDNF increased the phosphorylation of STAT3, and both a specific inhibitor of STAT3 and lentiviral shRNA for STAT3 decreased their differentiation into astrocytes. Taken together, our findings suggest that GDNF facilitates astrogliogenesis from neural precursor cells in adult DG through activating STAT3 and that this action might indirectly affect neurogenesis.

  1. Photoreceptor precursors derived from three-dimensional embryonic stem cell cultures integrate and mature within adult degenerate retina.

    PubMed

    Gonzalez-Cordero, Anai; West, Emma L; Pearson, Rachael A; Duran, Yanai; Carvalho, Livia S; Chu, Colin J; Naeem, Arifa; Blackford, Samuel J I; Georgiadis, Anastasios; Lakowski, Jorn; Hubank, Mike; Smith, Alexander J; Bainbridge, James W B; Sowden, Jane C; Ali, Robin R

    2013-08-01

    Irreversible blindness caused by loss of photoreceptors may be amenable to cell therapy. We previously demonstrated retinal repair and restoration of vision through transplantation of photoreceptor precursors obtained from postnatal retinas into visually impaired adult mice. Considerable progress has been made in differentiating embryonic stem cells (ESCs) in vitro toward photoreceptor lineages. However, the capability of ESC-derived photoreceptors to integrate after transplantation has not been demonstrated unequivocally. Here, to isolate photoreceptor precursors fit for transplantation, we adapted a recently reported three-dimensional (3D) differentiation protocol that generates neuroretina from mouse ESCs. We show that rod precursors derived by this protocol and selected via a GFP reporter under the control of a Rhodopsin promoter integrate within degenerate retinas of adult mice and mature into outer segment-bearing photoreceptors. Notably, ESC-derived precursors at a developmental stage similar to postnatal days 4-8 integrate more efficiently compared with cells at other stages. This study shows conclusively that ESCs can provide a source of photoreceptors for retinal cell transplantation.

  2. Long-term, stable differentiation of human embryonic stem cell-derived neural precursors grafted into the adult mammalian neostriatum.

    PubMed

    Nasonkin, Igor; Mahairaki, Vasiliki; Xu, Leyan; Hatfield, Glen; Cummings, Brian J; Eberhart, Charles; Ryugo, David K; Maric, Dragan; Bar, Eli; Koliatsos, Vassilis E

    2009-10-01

    Stem cell grafts have been advocated as experimental treatments for neurological diseases by virtue of their ability to offer trophic support for injured neurons and, theoretically, to replace dead neurons. Human embryonic stem cells (HESCs) are a rich source of neural precursors (NPs) for grafting, but have been questioned for their tendency to form tumors. Here we studied the ability of HESC-derived NP grafts optimized for cell number and differentiation stage prior to transplantation, to survive and stably differentiate and integrate in the basal forebrain (neostriatum) of young adult nude rats over long periods of time (6 months). NPs were derived from adherent monolayer cultures of HESCs exposed to noggin. After transplantation, NPs showed a drastic reduction in mitotic activity and an avid differentiation into neurons that projected via major white matter tracts to a variety of forebrain targets. A third of NP-derived neurons expressed the basal forebrain-neostriatal marker dopamine-regulated and cyclic AMP-regulated phosphoprotein. Graft-derived neurons formed mature synapses with host postsynaptic structures, including dendrite shafts and spines. NPs inoculated in white matter tracts showed a tendency toward glial (primarily astrocytic) differentiation, whereas NPs inoculated in the ventricular epithelium persisted as nestin(+) precursors. Our findings demonstrate the long-term ability of noggin-derived human NPs to structurally integrate tumor-free into the mature mammalian forebrain, while maintaining some cell fate plasticity that is strongly influenced by particular central nervous system (CNS) niches.

  3. Functional and phenotypic differences of pure populations of stem cell-derived astrocytes and neuronal precursor cells.

    PubMed

    Kleiderman, Susanne; Sá, João V; Teixeira, Ana P; Brito, Catarina; Gutbier, Simon; Evje, Lars G; Hadera, Mussie G; Glaab, Enrico; Henry, Margit; Sachinidis, Agapios; Alves, Paula M; Sonnewald, Ursula; Leist, Marcel

    2016-05-01

    Availability of homogeneous astrocyte populations would facilitate research concerning cell plasticity (metabolic and transcriptional adaptations; innate immune responses) and cell cycle reactivation. Current protocols to prepare astrocyte cultures differ in their final content of immature precursor cells, preactivated cells or entirely different cell types. A new method taking care of all these issues would improve research on astrocyte functions. We found here that the exposure of a defined population of pluripotent stem cell-derived neural stem cells (NSC) to BMP4 results in pure, nonproliferating astrocyte cultures within 24-48 h. These murine astrocytes generated from embryonic stem cells (mAGES) expressed the positive markers GFAP, aquaporin 4 and GLT-1, supported neuronal function, and acquired innate immune functions such as the response to tumor necrosis factor and interleukin 1. The protocol was applicable to several normal or disease-prone pluripotent cell lines, and the corresponding mAGES all exited the cell cycle and lost most of their nestin expression, in contrast to astrocytes generated by serum-addition or obtained as primary cultures. Comparative gene expression analysis of mAGES and NSC allowed quantification of differences between the two cell types and a definition of an improved marker set to define astrocytes. Inclusion of several published data sets in this transcriptome comparison revealed the similarity of mAGES with cortical astrocytes in vivo. Metabolic analysis of homogeneous NSC and astrocyte populations revealed distinct neurochemical features: both cell types synthesized glutamine and citrate, but only mature astrocytes released these metabolites. Thus, the homogeneous cultures allowed an improved definition of NSC and astrocyte features. © 2015 Wiley Periodicals, Inc.

  4. A Subpopulation of Smooth Muscle Cells, Derived from Melanocyte-Competent Precursors, Prevents Patent Ductus Arteriosus

    PubMed Central

    Puig, Isabel; Champeval, Delphine; Kumasaka, Mayuko; Belloir, Elodie; Bonaventure, Jacky; Mark, Manuel; Yamamoto, Hiroaki; Taketo, Mark M.; Choquet, Philippe; Etchevers, Heather C.; Beermann, Friedrich; Delmas, Véronique; Monassier, Laurent; Larue, Lionel

    2013-01-01

    Background Patent ductus arteriosus is a life-threatening condition frequent in premature newborns but also present in some term infants. Current mouse models of this malformation generally lead to perinatal death, not reproducing the full phenotypic spectrum in humans, in whom genetic inheritance appears complex. The ductus arteriosus (DA), a temporary fetal vessel that bypasses the lungs by shunting the aortic arch to the pulmonary artery, is constituted by smooth muscle cells of distinct origins (SMC1 and SMC2) and many fewer melanocytes. To understand novel mechanisms preventing DA closure at birth, we evaluated the importance of cell fate specification in SMC that form the DA during embryonic development. Upon specific Tyr::Cre-driven activation of Wnt/β-catenin signaling at the time of cell fate specification, melanocytes replaced the SMC2 population of the DA, suggesting that SMC2 and melanocytes have a common precursor. The number of SMC1 in the DA remained similar to that in controls, but insufficient to allow full DA closure at birth. Thus, there was no cellular compensation by SMC1 for the loss of SMC2. Mice in which only melanocytes were genetically ablated after specification from their potential common precursor with SMC2, demonstrated that differentiated melanocytes themselves do not affect DA closure. Loss of the SMC2 population, independent of the presence of melanocytes, is therefore a cause of patent ductus arteriosus and premature death in the first months of life. Our results indicate that patent ductus arteriosus can result from the insufficient differentiation, proliferation, or contractility of a specific smooth muscle subpopulation that shares a common neural crest precursor with cardiovascular melanocytes. PMID:23382837

  5. Non-PGM cathode catalysts for fuel cell application derived from heat treated heteroatomic amines precursors

    DOEpatents

    Serov, Alexey; Halevi, Barr; Artyushkova, Kateryna; Atanassov, Plamen B; Martinez, Ulises A

    2017-04-25

    A method of preparing M-N--C catalysts utilizing a sacrificial support approach and inexpensive and readily available polymer precursors as the source of nitrogen and carbon is disclosed. Exemplary polymer precursors include non-porphyrin precursors with no initial catalytic activity. Examples of suitable non-catalytic non-porphyrin precursors include, but are not necessarily limited to low molecular weight precursors that form complexes with iron such as 4-aminoantipirine, phenylenediamine, hydroxysuccinimide, ethanolamine, and the like.

  6. The effect of magnetic nanoparticles on neuronal differentiation of induced pluripotent stem cell-derived neural precursors

    PubMed Central

    Jiráková, Klára; Šeneklová, Monika; Jirák, Daniel; Turnovcová, Karolína; Vosmanská, Magda; Babič, Michal; Horák, Daniel; Veverka, Pavel; Jendelová, Pavla

    2016-01-01

    Introduction Magnetic resonance (MR) imaging is suitable for noninvasive long-term tracking. We labeled human induced pluripotent stem cell-derived neural precursors (iPSC-NPs) with two types of iron-based nanoparticles, silica-coated cobalt zinc ferrite nanoparticles (CZF) and poly-l-lysine-coated iron oxide superparamagnetic nanoparticles (PLL-coated γ-Fe2O3) and studied their effect on proliferation and neuronal differentiation. Materials and methods We investigated the effect of these two contrast agents on neural precursor cell proliferation and differentiation capability. We further defined the intracellular localization and labeling efficiency and analyzed labeled cells by MR. Results Cell proliferation was not affected by PLL-coated γ-Fe2O3 but was slowed down in cells labeled with CZF. Labeling efficiency, iron content and relaxation rates measured by MR were lower in cells labeled with CZF when compared to PLL-coated γ-Fe2O3. Cytoplasmic localization of both types of nanoparticles was confirmed by transmission electron microscopy. Flow cytometry and immunocytochemical analysis of specific markers expressed during neuronal differentiation did not show any significant differences between unlabeled cells or cells labeled with both magnetic nanoparticles. Conclusion Our results show that cells labeled with PLL-coated γ-Fe2O3 are suitable for MR detection, did not affect the differentiation potential of iPSC-NPs and are suitable for in vivo cell therapies in experimental models of central nervous system disorders. PMID:27920532

  7. The effect of magnetic nanoparticles on neuronal differentiation of induced pluripotent stem cell-derived neural precursors.

    PubMed

    Jiráková, Klára; Šeneklová, Monika; Jirák, Daniel; Turnovcová, Karolína; Vosmanská, Magda; Babič, Michal; Horák, Daniel; Veverka, Pavel; Jendelová, Pavla

    Magnetic resonance (MR) imaging is suitable for noninvasive long-term tracking. We labeled human induced pluripotent stem cell-derived neural precursors (iPSC-NPs) with two types of iron-based nanoparticles, silica-coated cobalt zinc ferrite nanoparticles (CZF) and poly-l-lysine-coated iron oxide superparamagnetic nanoparticles (PLL-coated γ-Fe2O3) and studied their effect on proliferation and neuronal differentiation. We investigated the effect of these two contrast agents on neural precursor cell proliferation and differentiation capability. We further defined the intracellular localization and labeling efficiency and analyzed labeled cells by MR. Cell proliferation was not affected by PLL-coated γ-Fe2O3 but was slowed down in cells labeled with CZF. Labeling efficiency, iron content and relaxation rates measured by MR were lower in cells labeled with CZF when compared to PLL-coated γ-Fe2O3. Cytoplasmic localization of both types of nanoparticles was confirmed by transmission electron microscopy. Flow cytometry and immunocytochemical analysis of specific markers expressed during neuronal differentiation did not show any significant differences between unlabeled cells or cells labeled with both magnetic nanoparticles. Our results show that cells labeled with PLL-coated γ-Fe2O3 are suitable for MR detection, did not affect the differentiation potential of iPSC-NPs and are suitable for in vivo cell therapies in experimental models of central nervous system disorders.

  8. Markers of pluripotency and differentiation in human neural precursor cells derived from embryonic stem cells and CNS tissue.

    PubMed

    Sundberg, M; Andersson, P-H; Åkesson, E; Odeberg, J; Holmberg, L; Inzunza, J; Falci, S; Öhman, J; Suuronen, R; Skottman, H; Lehtimäki, K; Hovatta, O; Narkilahti, S; Sundström, E

    2011-01-01

    Cell transplantation therapies for central nervous system (CNS) deficits such as spinal cord injury (SCI) have been shown to be effective in several animal models. One cell type that has been transplanted is neural precursor cells (NPCs), for which there are several possible sources. We have studied NPCs derived from human embryonic stem cells (hESCs) and human fetal CNS tissue (hfNPCs), cultured as neurospheres, and the expression of pluripotency and neural genes during neural induction and in vitro differentiation. mRNA for the pluripotency markers Nanog, Oct-4, Gdf3, and DNMT3b were downregulated during neural differentiation of hESCs. mRNA for these markers was found in nonpluripotent hfNPC at higher levels compared to hESC-NPCs. However, Oct-4 protein was found in hESC-NPCs after 8 weeks of culture, but not in hfNPCs. Similarly, SSEA-4 and CD326 were only found in hESC-NPCs. NPCs from both sources differentiated as expected to cells with typical features of neurons and astrocytes. The expressions of neuronal markers in hESC-NPCs were affected by the composition of cell culture medium, while this did not affect hfNPCs. Transplantation of hESC-NPC or hfNPC neurospheres into immunodeficient mouse testis or subcutaneous tissue did not result in tumor formation. In contrast, typical teratomas appeared in all animals after transplantation of hESC-NPCs to injured or noninjured spinal cords of immunodeficient rats. Our data show that transplantation to the subcutaneous tissue or the testes of immunodeficient mice is not a reliable method for evaluation of the tumor risk of remaining pluripotent cells in grafts. © 2011 Cognizant Comm. Corp.

  9. MEK Inhibitors Reverse Growth of Embryonal Brain Tumors Derived from Oligoneural Precursor Cells.

    PubMed

    Modzelewska, Katarzyna; Boer, Elena F; Mosbruger, Timothy L; Picard, Daniel; Anderson, Daniela; Miles, Rodney R; Kroll, Mitchell; Oslund, William; Pysher, Theodore J; Schiffman, Joshua D; Jensen, Randy; Jette, Cicely A; Huang, Annie; Stewart, Rodney A

    2016-10-25

    Malignant brain tumors are the leading cause of cancer-related deaths in children. Primitive neuroectodermal tumors of the CNS (CNS-PNETs) are particularly aggressive embryonal tumors of unknown cellular origin. Recent genomic studies have classified CNS-PNETs into molecularly distinct subgroups that promise to improve diagnosis and treatment; however, the lack of cell- or animal-based models for these subgroups prevents testing of rationally designed therapies. Here, we show that a subset of CNS-PNETs co-express oligoneural precursor cell (OPC) markers OLIG2 and SOX10 with coincident activation of the RAS/MAPK (mitogen-activated protein kinase) pathway. Modeling NRAS activation in embryonic OPCs generated malignant brain tumors in zebrafish that closely mimic the human oligoneural/NB-FOXR2 CNS-PNET subgroup by histology and comparative oncogenomics. The zebrafish CNS-PNET model was used to show that MEK inhibitors selectively eliminate Olig2(+)/Sox10(+) CNS-PNET tumors in vivo without impacting normal brain development. Thus, MEK inhibitors represent a promising rationally designed therapy for children afflicted with oligoneural/NB-FOXR2 CNS-PNETs.

  10. Presence of osteoclast precursor cells during ex vivo expansion of bone marrow-derived mesenchymal stem cells for autologous use in cell therapy.

    PubMed

    Allers, Carolina; Lasala, Gabriel P; Minguell, José J

    2014-04-01

    To obtain a cell product competent for clinical use in terms of cell dose and biologic properties, bone marrow-derived mesenchymal stem cells (MSCs) must be expanded ex vivo. A retrospective analysis was performed of records of 76 autologous MSC products used in phase I or II clinical studies performed in a cohort of cardiovascular patients. In all cases, native MSCs present in patient bone marrow aspirates were separated and expanded ex vivo. The cell products were classified in two groups (A and B), according to biologic properties and expansion time (ex vivo passages) to reach the protocol-established cell dose. In group A, the population of adherent cells obtained during the expansion period (2 ± 1 passages) was composed entirely of MSCs and met the requirements of cell number and biologic features as established in the respective clinical protocol. In group B, in addition to MSCs, we observed during expansion a high proportion of ancillary cells, characterized as osteoclast precursor cells. In this case, although the biologic properties of the resulting MSC product were not affected, the yield of MSCs was significantly lower. The expansion cycles had to be increased (3 ± 1 passages). These results suggest that the presence of osteoclast precursor cells in bone marrow aspirates may impose a limit for the proper clinical use of ex vivo expanded autologous bone marrow-derived MSCs. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  11. Programming pluripotent precursor cells derived from Xenopus embryos to generate specific tissues and organs.

    PubMed

    Borchers, Annette; Pieler, Tomas

    2010-11-18

    Xenopus embryos provide a rich source of pluripotent cells that can be differentiated into functional organs. Since the molecular principles of vertebrate organogenesis appear to be conserved between Xenopus and mammals, this system can provide useful guidelines for the directional manipulation of human embryonic stem cells. Pluripotent Xenopus cells can be easily isolated from the animal pole of blastula stage Xenopus embryos. These so called "animal cap" cells represent prospective ectodermal cells, but give rise to endodermal, mesodermal and neuro-ectodermal derivatives if treated with the appropriate factors. These factors include evolutionary conserved modulators of the key developmental signal transduction pathways that can be supplied either by mRNA microinjection or direct application of recombinant proteins. This relatively simple system has added to our understanding of pancreas, liver, kidney, eye and heart development. In particular, recent studies have used animal cap cells to generate ectopic eyes and hearts, setting the stage for future work aimed at programming pluripotent cells for regenerative medicine.

  12. IAP-Based Cell Sorting Results in Homogeneous Transplantable Dopaminergic Precursor Cells Derived from Human Pluripotent Stem Cells.

    PubMed

    Lehnen, Daniela; Barral, Serena; Cardoso, Tiago; Grealish, Shane; Heuer, Andreas; Smiyakin, Andrej; Kirkeby, Agnete; Kollet, Jutta; Cremer, Harold; Parmar, Malin; Bosio, Andreas; Knöbel, Sebastian

    2017-09-19

    Human pluripotent stem cell (hPSC)-derived mesencephalic dopaminergic (mesDA) neurons can relieve motor deficits in animal models of Parkinson's disease (PD). Clinical translation of differentiation protocols requires standardization of production procedures, and surface-marker-based cell sorting is considered instrumental for reproducible generation of defined cell products. Here, we demonstrate that integrin-associated protein (IAP) is a cell surface marker suitable for enrichment of hPSC-derived mesDA progenitor cells. Immunomagnetically sorted IAP(+) mesDA progenitors showed increased expression of ventral midbrain floor plate markers, lacked expression of pluripotency markers, and differentiated into mature dopaminergic (DA) neurons in vitro. Intrastriatal transplantation of IAP(+) cells sorted at day 16 of differentiation in a rat model of PD resulted in functional recovery. Grafts from sorted IAP(+) mesDA progenitors were more homogeneous in size and DA neuron density. Thus, we suggest IAP-based sorting for reproducible prospective enrichment of mesDA progenitor cells in clinical cell replacement strategies. Copyright © 2017 Miltenyi Biotec GmbH. Published by Elsevier Inc. All rights reserved.

  13. Riluzole enhances expression of brain-derived neurotrophic factor with consequent proliferation of granule precursor cells in the rat hippocampus.

    PubMed

    Katoh-Semba, Ritsuko; Asano, Tomiko; Ueda, Hiroshi; Morishita, Rika; Takeuchi, Ikuo K; Inaguma, Yutaka; Kato, Kanefusa

    2002-08-01

    The dentate gyrus of the hippocampus, generating new cells throughout life, is essential for normal recognition memory performance. Reduction of brain-derived neurotrophic factor (BDNF) in this structure impairs its functions. To elucidate the association between BDNF levels and hippocampal neurogenesis, we first conducted a search for compounds that stimulate endogenous BDNF production in hippocampal granule neurons. Among ion channel modulators tested, riluzole, a neuroprotective agent with anticonvulsant properties that is approved for treatment of amyotrophic lateral sclerosis, was highly effective as a single dose by an intraperitoneal injection, causing a rise in BDNF localized in dentate granule neurons, the hilus, and the stratum radiatum of the CA3 region. Repeated, but not single, injections resulted in prolonged elevation of hippocampal BDNF and were associated with increased numbers of newly generated cells in the granule cell layer. This appeared due to promoted proliferation rather than survival of precursor cells, many of which differentiated into neurons. Intraventricular administration of BDNF-specific antibodies blocked such riluzole effects, suggesting that BDNF increase is necessary for the promotion of precursor proliferation. Our results suggest the basis for a new strategy for treatment of memory dysfunction.

  14. Neural Growth Factor Stimulates Proliferation of Spinal Cord Derived-Neural Precursor/Stem Cells

    PubMed Central

    Han, Youngmin

    2016-01-01

    Objective Recently, regenerative therapies have been used in clinical trials (heart, cartilage, skeletal). We don't make use of these treatments to spinal cord injury (SCI) patients yet, but regenerative therapies are rising interest in recent study about SCI. Neural precursor/stem cell (NPSC) proliferation is a significant event in functional recovery of the central nervous system (CNS). However, brain NPSCs and spinal cord NPSCs (SC-NPSCs) have many differences including gene expression and proliferation. The purpose of this study was to investigate the influence of neural growth factor (NGF) on the proliferation of SC-NPSCs. Methods NPSCs (2×104) were suspended in 100 µL of neurobasal medium containing NGF-7S (Sigma-Aldrich) and cultured in a 96-well plate for 12 days. NPSC proliferation was analyzed five times for either concentration of NGF (0.02 and 2 ng/mL). Sixteen rats after SCI were randomly allocated into two groups. In group 1 (SCI-vehicle group, n=8), animals received 1.0 mL of the saline vehicle solution. In group 2 (SCI-NGF group, n=8), the animals received single doses of NGF (Sigma-Aldrich). A dose of 0.02 ng/mL of NGF or normal saline as a vehicle control was intra-thecally injected daily at 24 hour intervals for 7 days. For Immunohistochemistry analysis, rats were sacrificed after one week and the spinal cords were obtained. Results The elevation of cell proliferation with 0.02 ng/mL NGF was significant (p<0.05) but was not significant for 2 ng/mL NGF. The optical density was increased in the NGF 0.02 ng/mL group compared to the control group and NGF 2 ng/mL groups. The density of nestin in the SCI-NGF group was significantly increased over the SCI-vehicle group (p<0.05). High power microscopy revealed that the density of nestin in the SCI-NGF group was significantly increased over the SCI-vehicle group. Conclusion SC-NPSC proliferation is an important pathway in the functional recovery of SCI. NGF enhances SC-NPSC proliferation in vitro and in

  15. Transplantation of Photoreceptor Precursors Isolated via a Cell Surface Biomarker Panel From Embryonic Stem Cell-Derived Self-Forming Retina.

    PubMed

    Lakowski, Jorn; Gonzalez-Cordero, Anai; West, Emma L; Han, Ya-Ting; Welby, Emily; Naeem, Arifa; Blackford, Samuel J I; Bainbridge, James W B; Pearson, Rachael A; Ali, Robin R; Sowden, Jane C

    2015-08-01

    Loss of photoreceptors due to retinal degeneration is a major cause of untreatable blindness. Cell replacement therapy, using pluripotent stem cell-derived photoreceptor cells, may be a feasible future treatment. Achieving safe and effective cell replacement is critically dependent on the stringent selection and purification of optimal cells for transplantation. Previously, we demonstrated effective transplantation of post-mitotic photoreceptor precursor cells labelled by fluorescent reporter genes. As genetically labelled cells are not desirable for therapy, here we developed a surface biomarker cell selection strategy for application to complex pluripotent stem cell differentiation cultures. We show that a five cell surface biomarker panel CD73(+)CD24(+)CD133(+)CD47(+)CD15(-) facilitates the isolation of photoreceptor precursors from three-dimensional self-forming retina differentiated from mouse embryonic stem cells. Importantly, stem cell-derived cells isolated using the biomarker panel successfully integrate and mature into new rod photoreceptors in the adult mouse retinae after subretinal transplantation. Conversely, unsorted or negatively selected cells do not give rise to newly integrated rods after transplantation. The biomarker panel also removes detrimental proliferating cells prior to transplantation. Notably, we demonstrate how expression of the biomarker panel is conserved in the human retina and propose that a similar selection strategy will facilitate isolation of human transplantation-competent cells for therapeutic application.

  16. Region-Specific Integration of Embryonic Stem Cell-Derived Neuronal Precursors into a Pre-Existing Neuronal Circuit

    PubMed Central

    Neuser, Franziska; Polack, Martin; Annaheim, Christine; Tucker, Kerry L.; Korte, Martin

    2013-01-01

    Enduring reorganization is accepted as a fundamental process of adult neural plasticity. The most dramatic example of this reorganization is the birth and continuously occurring incorporation of new neurons into the pre-existing network of the adult mammalian hippocampus. Based on this phenomenon we transplanted murine embryonic stem (ES)-cell derived neuronal precursors (ESNPs) into murine organotypic hippocampal slice cultures (OHC) and examined their integration. Using a precise quantitative morphological analysis combined with a detailed electrophysiology, we show a region-specific morphological integration of transplanted ESNPs into different subfields of the hippocampal tissue, resulting in pyramidal neuron-like embryonic stem cell-derived neurons (ESNs) in the Cornu Ammonis (CA1 and CA3) and granule neuron-like ESNs in the dentate gyrus (DG), respectively. Subregion specific structural maturation was accompanied by the development of dendritic spines and the generation of excitatory postsynaptic currents (EPSCs). This cell type specific development does not depend upon NMDA-receptor-dependent synaptic transmission. The presented integration approach was further used to determine the cell-autonomous function of the pan-neurotrophin receptor p75 (P75NTR), as a possible negative regulator of ESN integration. By this means we used p75NTR-deficient ESNPs to study their integration into a WT organotypic environment. We show here that p75NTR is not necessary for integration per se but plays a suppressing role in dendritic development. PMID:23840491

  17. Polysialic acid modification of the synaptic cell adhesion molecule SynCAM 1 in human embryonic stem cell-derived oligodendrocyte precursor cells.

    PubMed

    Werneburg, Sebastian; Buettner, Falk F R; Mühlenhoff, Martina; Hildebrandt, Herbert

    2015-05-01

    Oligodendrocyte precursor cells (OPCs) are the progenitors of myelinating oligodendrocytes in brain development and repair. Successful myelination depends on the control of adhesiveness during OPC migration and axon contact formation. The decoration of cell surface proteins with the glycan polysialic acid (polySia) is a key regulatory element of OPC interactions during development and under pathological conditions. By far the major protein carrier of polySia is the neural cell adhesion molecule NCAM, but recently, polysialylation of the synaptic cell adhesion molecule SynCAM 1 has been detected in the developing mouse brain. In mice, polySia-SynCAM 1 is associated with cells expressing NG2, a marker of a heterogeneous precursor cell population, which is the primary source for oligodendrocytes in development and myelin repair but can also give rise to astrocytes and possibly neurons. It is not yet clear if polySia-SynCAM 1 is expressed by OPCs and its occurrence in humans is elusive. By generating uniform human embryonic stem cell-derived OPC cultures, we demonstrate that polySia is present on human OPCs but down-regulated during differentiation into myelin basic protein-positive oligodendrocytes. PolySia on NCAM resides on the isoforms NCAM-180 and NCAM-140, and SynCAM 1 is identified as a novel polySia acceptor in human OPCs.

  18. Hair Follicle and Sebaceous Gland De Novo Regeneration With Cultured Epidermal Stem Cells and Skin-Derived Precursors.

    PubMed

    Wang, Xiaoxiao; Wang, Xusheng; Liu, Jianjun; Cai, Ting; Guo, Ling; Wang, Shujuan; Wang, Jinmei; Cao, Yanpei; Ge, Jianfeng; Jiang, Yuyang; Tredget, Edward E; Cao, Mengjun; Wu, Yaojiong

    2016-12-01

    : Stem cell-based organ regeneration is purported to enable the replacement of impaired organs in the foreseeable future. Here, we demonstrated that a combination of cultured epidermal stem cells (Epi-SCs) derived from the epidermis and skin-derived precursors (SKPs) was capable of reconstituting functional hair follicles and sebaceous glands (SG). When Epi-SCs and SKPs were mixed in a hydrogel and implanted into an excisional wound in nude mice, the Epi-SCs formed de novo epidermis along with hair follicles, and SKPs contributed to dermal papilla in the neogenic hair follicles. Notably, a combination of culture-expanded Epi-SCs and SKPs derived from the adult human scalp were sufficient to generate hair follicles and hair. Bone morphogenetic protein 4, but not Wnts, sustained the expression of alkaline phosphatase in SKPs in vitro and the hair follicle-inductive property in vivo when SKPs were engrafted with neonatal epidermal cells into excisional wounds. In addition, Epi-SCs were capable of differentiating into sebocytes and formed de novo SGs, which excreted lipids as do normal SGs. Thus our results indicate that cultured Epi-SCs and SKPs are sufficient to generate de novo hair follicles and SGs, implying great potential to develop novel bioengineered skin substitutes with appendage genesis capacity. In postpartum humans, skin appendages lost in injury are not regenerated, despite the considerable achievement made in skin bioengineering. In this study, transplantation of a combination of culture-expanded epidermal stem cells and skin-derived progenitors from mice and adult humans led to de novo regeneration of functional hair follicles and sebaceous glands. The data provide transferable knowledge for the development of novel bioengineered skin substitutes with epidermal appendage regeneration capacity. ©AlphaMed Press.

  19. Adult skin-derived precursor Schwann cells exhibit superior myelination and regeneration supportive properties compared to chronically denervated nerve-derived Schwann cells.

    PubMed

    Kumar, Ranjan; Sinha, Sarthak; Hagner, Andrew; Stykel, Morgan; Raharjo, Eko; Singh, Karun K; Midha, Rajiv; Biernaskie, Jeff

    2016-04-01

    Functional outcomes following delayed peripheral nerve repair are poor. Schwann cells (SCs) play key roles in supporting axonal regeneration and remyelination following nerve injury, thus understanding the impact of chronic denervation on SC function is critical toward developing therapies to enhance regeneration. To improve our understanding of SC function following acute versus chronic-denervation, we performed functional assays of SCs from adult rodent sciatic nerve with acute- (Day 5 post) or chronic-denervation (Day 56 post), versus embryonic nerves. We also compared Schwann cells derived from adult skin-derived precursors (aSKP-SCs) as an accessible, autologous alternative to supplement the distal (denervated) nerve. We found that acutely-injured SCs and aSKP-SCs exhibited superior proliferative capacity, promotion of neurite outgrowth and myelination of axons, both in vitro and following transplant into a sciatic nerve crush injury model, while chronically-denervated SCs were severely impaired. Acute injury caused re-activation of transcription factors associated with an immature and pro-myelinating SC state (Oct-6, cJun, Sox2, AP2α, cadherin-19), but was diminished with prolonged denervation in vivo and could not be rescued following expansion in vitro suggesting that this is a permanent deficiency. Interestingly, aSKP-SCs closely resembled acutely injured and embryonic SCs, exhibiting elevated expression of these same transcription factors. In summary, prolonged denervation resulted in SC deficiency in several functional parameters that may contribute to impaired regeneration. In contrast, aSKP-SCs closely resemble the regenerative attributes ascribed to acutely-denervated or embryonic SCs emphasizing their potential as an accessible and autologous source of glia cells to enhance nerve regeneration, particularly following delays to surgical repair.

  20. Precursor N-cadherin mediates glial cell line-derived neurotrophic factor-promoted human malignant glioma

    PubMed Central

    Zhu, Shuang; Zhang, Baole; Qin, Yuxia; Yao, Ruiqin; Zhou, Hao; Gao, Dian Shuai

    2017-01-01

    As the most prevalent primary brain tumor, gliomas are highly metastatic, invasive and are characteristic of high levels of glial cell-line derived neurotrophic factor (GDNF). GDNF is an important factor for invasive glioma cell growth; however, the underlying mechanism involved is unclear. In this study, we affirm a significantly higher expression of the precursor of N-cadherin (proN-cadherin) in most gliomas compared with normal brain tissues. Our findings reveal that GDNF interacts with the extracellular domain of proN-cadherin, which suggests that proN-cadherin mediates GDNF-induced glioma cell migration and invasion. We hypothesize that proN-cadherin might cause homotypic adhesion loss within neighboring cells and at the same time promote heterotypic adhesion within the extracellular matrix (ECM) through a certain mechanism. This study also demonstrates that the interaction between GDNF and proN-cadherin activates specific intracellular signaling pathways; furthermore, GDNF promoted the secretion of matrix metalloproteinase-9 (MMP-9), which degrades the ECM via proN-cadherin. To reach the future goal of developing novel therapies of glioma, this study, reveals a unique mechanism of glioma cell migration and invasion. PMID:28212546

  1. Enhanced Expansion and Sustained Inductive Function of Skin‐Derived Precursor Cells in Computer‐Controlled Stirred Suspension Bioreactors

    PubMed Central

    Agabalyan, Natacha A.; Borys, Breanna S.; Sparks, Holly D.; Boon, Kathryn; Raharjo, Eko W.; Abbasi, Sepideh; Kallos, Michael S.

    2016-01-01

    Abstract Endogenous dermal stem cells (DSCs) reside in the adult hair follicle mesenchyme and can be isolated and grown in vitro as self‐renewing colonies called skin‐derived precursors (SKPs). Following transplantation into skin, SKPs can generate new dermis and reconstitute the dermal papilla and connective tissue sheath, suggesting they could have important therapeutic value for the treatment of skin disease (alopecia) or injury. Controlled cell culture processes must be developed to efficiently and safely generate sufficient stem cell numbers for clinical use. Compared with static culture, stirred‐suspension bioreactors generated fivefold greater expansion of viable SKPs. SKPs from each condition were able to repopulate the dermal stem cell niche within established hair follicles. Both conditions were also capable of inducing de novo hair follicle formation and exhibited bipotency, reconstituting the dermal papilla and connective tissue sheath, although the efficiency was significantly reduced in bioreactor‐expanded SKPs compared with static conditions. We conclude that automated bioreactor processing could be used to efficiently generate large numbers of autologous DSCs while maintaining their inherent regenerative function. Stem Cells Translational Medicine 2017;6:434–443 PMID:28191777

  2. Precursor N-cadherin mediates glial cell line-derived neurotrophic factor-promoted human malignant glioma.

    PubMed

    Xiong, Ye; Liu, Liyun; Zhu, Shuang; Zhang, Baole; Qin, Yuxia; Yao, Ruiqin; Zhou, Hao; Gao, Dian Shuai

    2017-02-12

    As the most prevalent primary brain tumor, gliomas are highly metastatic, invasive and are characteristic of high levels of glial cell-line derived neurotrophic factor (GDNF). GDNF is an important factor for invasive glioma cell growth; however, the underlying mechanism involved is unclear. In this study, we affirm a significantly higher expression of the precursor of N-cadherin (proN-cadherin) in most gliomas compared with normal brain tissues. Our findings reveal that GDNF interacts with the extracellular domain of proN-cadherin, which suggests that proN-cadherin mediates GDNF-induced glioma cell migration and invasion. We hypothesize that proN-cadherin might cause homotypic adhesion loss within neighboring cells and at the same time promote heterotypic adhesion within the extracellular matrix (ECM) through a certain mechanism. This study also demonstrates that the interaction between GDNF and proN-cadherin activates specific intracellular signaling pathways; furthermore, GDNF promoted the secretion of matrix metalloproteinase-9 (MMP-9), which degrades the ECM via proN-cadherin. To reach the future goal of developing novel therapies of glioma, this study, reveals a unique mechanism of glioma cell migration and invasion.

  3. Nanofiber Matrices Promote the Neuronal Differentiation of Human Embryonic Stem Cell-Derived Neural Precursors In Vitro

    PubMed Central

    Lim, Shawn H.; Christopherson, Gregory T.; Xu, Leyan; Nasonkin, Igor; Yu, Christopher; Mao, Hai-Quan; Koliatsos, Vassilis E.

    2011-01-01

    The potential of human embryonic stem (ES) cells as experimental therapies for neuronal replacement has recently received considerable attention. In view of the organization of the mature nervous system into distinct neural circuits, key challenges of such therapies are the directed differentiation of human ES cell-derived neural precursors (NPs) into specific neuronal types and the directional growth of axons along specified trajectories. In the present study, we cultured human NPs derived from the NIH-approved ES line BGO1 on polycaprolactone fiber matrices of different diameter (i.e., nanofibers and microfibers) and orientation (i.e., aligned and random); fibers were coated with poly-L-ornithine/laminin to mimic the extracellular matrix and support the adhesion, viability, and differentiation of NPs. On aligned fibrous meshes, human NPs adopt polarized cell morphology with processes extending along the axis of the fiber, whereas NPs on plain tissue culture surfaces or random fiber substrates form nonpolarized neurite networks. Under differentiation conditions, human NPs cultured on aligned fibrous substrates show a higher rate of neuronal differentiation than other matrices; 62% and 86% of NPs become TUJ1 (+) early neurons on aligned micro- and nanofibers, respectively, whereas only 32% and 27% of NPs acquire the same fate on random micro- and nanofibers. Metabolic cell activity/viability studies reveal that fiber alignment and diameter also have an effect on NP viability, but only in the presence of mitogens. Our findings demonstrate that fibrous substrates serve as an artificial extracellular matrix and provide a microenviroment that influences key aspects of the neuronal differentiation of ES-derived NPs. PMID:20973749

  4. Functional effects of cannabinoids during dopaminergic specification of human neural precursors derived from induced pluripotent stem cells.

    PubMed

    Stanslowsky, Nancy; Jahn, Kirsten; Venneri, Anna; Naujock, Maximilian; Haase, Alexandra; Martin, Ulrich; Frieling, Helge; Wegner, Florian

    2016-03-30

    Among adolescents cannabis is one of the most widely used illicit drugs. In adolescence brain development continues, characterized by neuronal maturation and synaptic plasticity. The endocannabinoid system plays an important role during brain development by modulating neuronal function and neurogenesis. Changes in endocannabinoid signaling by Δ(9) -tetrahydrocannabinol (THC), the psychoactive component of cannabis, might therefore lead to neurobiological changes influencing brain function and behavior. We investigated the functional maturation and dopaminergic specification of human cord blood-derived induced pluripotent stem cell (hCBiPSC)-derived small molecule neural precursor cells (smNPCs) after cultivation with the endogenous cannabinoid anandamide (AEA) and the exogenous THC, both potent agonists at the cannabinoid 1 receptor (CB1 R). Higher dosages of 10-μM AEA or THC significantly decreased functionality of neurons, indicated by reduced ion currents and synaptic activity. A lower concentration of 1-μM THC had no marked effect on neuronal and dopaminergic maturation, while 1-μM AEA significantly enhanced the frequency of synaptic activity. As there were no significant effects on DNA methylation in promotor regions of genes important for neuronal function, these cannabinoid actions seem to be mediated by another than this epigenetic mechanism. Our data suggest that there are concentration-dependent actions of cannabinoids on neuronal function in vitro indicating neurotoxic, dysfunctional effects of 10-μM AEA and THC during human neurogenesis.

  5. Grafted Human iPS Cell-Derived Oligodendrocyte Precursor Cells Contribute to Robust Remyelination of Demyelinated Axons after Spinal Cord Injury.

    PubMed

    Kawabata, Soya; Takano, Morito; Numasawa-Kuroiwa, Yuko; Itakura, Go; Kobayashi, Yoshiomi; Nishiyama, Yuichiro; Sugai, Keiko; Nishimura, Soraya; Iwai, Hiroki; Isoda, Miho; Shibata, Shinsuke; Kohyama, Jun; Iwanami, Akio; Toyama, Yoshiaki; Matsumoto, Morio; Nakamura, Masaya; Okano, Hideyuki

    2016-01-12

    Murine- and human-induced pluripotent stem cell-derived neural stem/progenitor cells (iPSC-NS/PCs) promote functional recovery following transplantation into the injured spinal cord in rodents and primates. Although remyelination of spared demyelinated axons is a critical mechanism in the regeneration of the injured spinal cord, human iPSC-NS/PCs predominantly differentiate into neurons both in vitro and in vivo. We therefore took advantage of our recently developed protocol to obtain human-induced pluripotent stem cell-derived oligodendrocyte precursor cell-enriched neural stem/progenitor cells and report the benefits of transplanting these cells in a spinal cord injury (SCI) model. We describe how this approach contributes to the robust remyelination of demyelinated axons and facilitates functional recovery after SCI. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Grafted Human iPS Cell-Derived Oligodendrocyte Precursor Cells Contribute to Robust Remyelination of Demyelinated Axons after Spinal Cord Injury

    PubMed Central

    Kawabata, Soya; Takano, Morito; Numasawa-Kuroiwa, Yuko; Itakura, Go; Kobayashi, Yoshiomi; Nishiyama, Yuichiro; Sugai, Keiko; Nishimura, Soraya; Iwai, Hiroki; Isoda, Miho; Shibata, Shinsuke; Kohyama, Jun; Iwanami, Akio; Toyama, Yoshiaki; Matsumoto, Morio; Nakamura, Masaya; Okano, Hideyuki

    2015-01-01

    Summary Murine- and human-induced pluripotent stem cell-derived neural stem/progenitor cells (iPSC-NS/PCs) promote functional recovery following transplantation into the injured spinal cord in rodents and primates. Although remyelination of spared demyelinated axons is a critical mechanism in the regeneration of the injured spinal cord, human iPSC-NS/PCs predominantly differentiate into neurons both in vitro and in vivo. We therefore took advantage of our recently developed protocol to obtain human-induced pluripotent stem cell-derived oligodendrocyte precursor cell-enriched neural stem/progenitor cells and report the benefits of transplanting these cells in a spinal cord injury (SCI) model. We describe how this approach contributes to the robust remyelination of demyelinated axons and facilitates functional recovery after SCI. PMID:26724902

  7. Derivation of Schwann cell precursors from neural crest cells resident in bone marrow for cell therapy to improve peripheral nerve regeneration.

    PubMed

    Shi, Haiyan; Gong, Yanpei; Qiang, Liang; Li, Xiaoli; Zhang, Shibo; Gao, Jiawen; Li, Kai; Ji, Ximeng; Tian, Ling; Gu, Xiaosong; Ding, Fei

    2016-05-01

    We have previously successfully enriched post-migratory neural crest cells (NCCs) from postnatal rat bone marrow (BM). These BM-NCCs possess glial and neuronal differentiating potential. Based on the neural crest origin of Schwann cells (SCs), in this study, we aimed at using a straightforward protocol to derive Schwann cell precursors (SCPs) from BM-NCCs. Several clonal subpopulations were isolated from BM-NCCs, displaying long-term proliferative capacity and maintaining the NCC identity. The BM-NCC clones could be induced to differentiate into SCs. In particular, clone N1 gave rise to a large and pure population of SCs. Clone N1-derived SCs demonstrated the myelinating capacity in their co-culture with primary dorsal root ganglion (DRG) neurons. The decreased expression of NCC-markers and increased expression of SC-markers were related to the differentiation state of clone N1-derived SCs. To investigate the repair-promoting effects of clone N1 on injured peripheral neurons in vitro and in vivo, on one hand, the oxygen glucose deprivation-injured DRG neurons were treated with clone N1-conditioned medium, improving the cell survival and axon growth of neurons; on the other hand, clone N1 or clone N1-derived SCs were respectively implanted to the crush sciatic nerve of rats, and clone N1 yielded the better outcome of nerve regeneration and function restoration than clone N1-derived SCs. Taken together, all the results collectively showed that clone N1 could be identified as SCPs, which might hold promise for cell therapy to improve peripheral nerve regeneration. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Brain-Derived Neurotrophic Factor Induces Cell Survival and the Migration of Murine Adult Hippocampal Precursor Cells During Differentiation In Vitro.

    PubMed

    Ortiz-López, Leonardo; Vega-Rivera, Nelly Maritza; Babu, Harish; Ramírez-Rodríguez, Gerardo Bernabé

    2017-01-01

    The generation of new neurons during adulthood involves local precursor cell migration and terminal differentiation in the dentate gyrus. These events are influenced by the hippocampal microenvironment. Brain-derived neurotrophic factor (BDNF) is relevant for hippocampal neuronal development and behavior. Interestingly, studies that have been performed in controlled in vitro systems that involve isolated precursor cells that were derived from the dentate gyrus (AHPCs) have shown that BDNF induces the activation of the TrkB receptor and, consequentially, might activate signaling pathways that favor survival and neuronal differentiation. Based on the fact that the cellular events of AHPCs that are induced by single factors can be studied in this controlled in vitro system, we investigated the ability of BDNF and the involvement of protein kinase C (PKC), as one of the TrkB-downstream activated signaling proteins, in the regulation of migration, here reflected by motility, of AHPCs. Precursor cells were cultured following a concentration-response curve (1-640 ng/ml) for 24 or 96 h. We found that BDNF favored cell survival without altering the viability under culture proliferative conditions of the AHPCs. Concomitantly, glial- and neuronal-differentiated precursor cells increased as a consequence of survival promoted by BDNF. Additionally, pharmacological approaches showed that BDNF (40 ng/ml)-induced migration of AHPCs was blocked with the compounds K252a and GF109203x, which prevent the activation of TrkB and PKC, respectively. The results indicate that in the in vitro migration of differentiated AHPCs it is involved the BDNF and TrkB cascade. Our results provide additional information about the mechanism by which BDNF impacts adult neurogenesis in the hippocampus.

  9. The granulin-epithelin precursor/PC-cell-derived growth factor is a growth factor for epithelial ovarian cancer.

    PubMed

    Jones, Monica Brown; Michener, Chad M; Blanchette, James O; Kuznetsov, Vladimir A; Raffeld, Mark; Serrero, Ginette; Emmert-Buck, Michael R; Petricoin, Emanuel F; Krizman, David B; Liotta, Lance A; Kohn, Elise C

    2003-01-01

    The role of growth factors in ovarian cancer development and progression is complex and multifactorial. We hypothesized that new growth factors may be identified through the molecular analysis of ovarian tumors as they exist in their native environment. RNA extracted from microdissected serous low malignant potential (LMP) and invasive ovarian tumors was used to construct cDNA libraries. A total of 7300 transcripts were randomly chosen for sequencing, and those transcripts were statistically evaluated. Reverse transcription-PCR and immunohistochemistry were used to validate the findings in tumor tissue samples. Ovarian cancer cell lines were used to test gene effects on monolayer growth, proliferative capacity, and density-independent growth. Analysis of the pooled library transcripts revealed 26 genes differentially expressed between LMP and invasive ovarian cancers. The granulin-epithelin precursor [GEP/PC-cell derived growth factor (PCDGF)] was expressed only in the invasive ovarian cancer libraries (P < 0.028) and was absent in the LMP libraries (0 of 2872 clones). All of the invasive tumor epithelia, 20% of the LMP tumor epithelia, and all of the stroma from both subsets expressed GEP by reverse transcription-PCR. Immunohistochemical staining for GEP was diffuse and cytosolic in invasive ovarian cancer tumor cells compared with occasional, punctate, and apical staining in LMP tumor epithelia. Antisense transfection of GEP into ovarian cancer cell lines resulted in down-regulation of GEP production, reduction in cell growth (P < 0.002), decrease in the S-phase fraction (P < 0.04), and loss of density-independent growth potential (P < 0.01). cDNA library preparation from microdissected tumor epithelium provided a selective advantage for the identification of growth factors for epithelial ovarian cancer. Differential granulin expression in tumor samples and the antiproliferative effects of its antisense down-regulation suggest that GEP may be a new autocrine

  10. Aligned Poly(ε-caprolactone) Nanofibers Guide the Orientation and Migration of Human Pluripotent Stem Cell-Derived Neurons, Astrocytes, and Oligodendrocyte Precursor Cells In Vitro.

    PubMed

    Hyysalo, Anu; Ristola, Mervi; Joki, Tiina; Honkanen, Mari; Vippola, Minnamari; Narkilahti, Susanna

    2017-03-15

    Stem cell transplantations for spinal cord injury (SCI) have been studied extensively for the past decade in order to replace the damaged tissue with human pluripotent stem cell (hPSC)-derived neural cells. Transplanted cells may, however, benefit from supporting and guiding structures or scaffolds in order to remain viable and integrate into the host tissue. Biomaterials can be used as supporting scaffolds, as they mimic the characteristics of the natural cellular environment. In this study, hPSC-derived neurons, astrocytes, and oligodendrocyte precursor cells (OPCs) are cultured on aligned poly(ε-caprolactone) nanofiber platforms, which guide cell orientation to resemble that of spinal cord in vivo. All cell types are shown to efficiently spread over the nanofiber platform and orient according to the fiber alignment. Human neurons and astrocytes require extracellular matrix molecule coating for the nanofibers, but OPCs grow on nanofibers without additional treatment. Furthermore, the nanofiber platform is combined with a 3D hydrogel scaffold with controlled thickness, and nanofiber-mediated orientation of hPSC-derived neurons is also demonstrated in a 3D environment. In this work, clinically relevant materials and substrates for nanofibers, fiber coatings, and hydrogel scaffolds are used and combined with cells suitable for developing functional cell grafts for SCI repair.

  11. Antigen receptor-redirected T cells derived from hematopoietic precursor cells lack expression of the endogenous TCR/CD3 receptor and exhibit specific antitumor capacities

    PubMed Central

    Van Caeneghem, Yasmine; De Munter, Stijn; Tieppo, Paola; Goetgeluk, Glenn; Weening, Karin; Verstichel, Greet; Bonte, Sarah; Taghon, Tom; Leclercq, Georges; Kerre, Tessa; Debets, Reno; Vermijlen, David; Abken, Hinrich; Vandekerckhove, Bart

    2017-01-01

    ABSTRACT Recent clinical studies indicate that adoptive T-cell therapy and especially chimeric antigen receptor (CAR) T-cell therapy is a very potent and potentially curative treatment for B-lineage hematologic malignancies. Currently, autologous peripheral blood T cells are used for adoptive T-cell therapy. Adoptive T cells derived from healthy allogeneic donors may have several advantages; however, the expected occurrence of graft versus host disease (GvHD) as a consequence of the diverse allogeneic T-cell receptor (TCR) repertoire expressed by these cells compromises this approach. Here, we generated T cells from cord blood hematopoietic progenitor cells (HPCs) that were transduced to express an antigen receptor (AR): either a CAR or a TCR with or without built-in CD28 co-stimulatory domains. These AR-transgenic HPCs were culture-expanded on an OP9-DL1 feeder layer and subsequently differentiated to CD5+CD7+ T-lineage precursors, to CD4+ CD8+ double positive cells and finally to mature AR+ T cells. The AR+ T cells were largely naive CD45RA+CD62L+ T cells. These T cells had mostly germline TCRα and TCRβ loci and therefore lacked surface-expressed CD3/TCRαβ complexes. The CD3− AR-transgenic cells were mono-specific, functional T cells as they displayed specific cytotoxic activity. Cytokine production, including IL-2, was prominent in those cells bearing ARs with built-in CD28 domains. Data sustain the concept that cord blood HPC derived, in vitro generated allogeneic CD3− AR+ T cells can be used to more effectively eliminate malignant cells, while at the same time limiting the occurrence of GvHD. PMID:28405508

  12. Effects of short-term exposure to sevoflurane on the survival, proliferation, apoptosis, and differentiation of neural precursor cells derived from human embryonic stem cells.

    PubMed

    Park, Jin-Woo; Lim, Mi-Sun; Ji, So Yeon; Cho, Myung Soo; Park, Seong-Joo; Han, Sung-Hee; Kim, Jin-Hee

    2017-09-14

    Data from animal experiments suggest that exposure to general anesthetics in early life inhibits neurogenesis and causes long-term memory deficit. Considering short operating times and the popularity of sevoflurane in pediatric anesthesia, it is important to verify the effects of short-period exposure to sevoflurane on the developing brain. We measured the effects of short-term exposure (2 h) to 3%, 6%, or 8% sevoflurane, the most commonly used anesthetic, on neural precursor cells derived from human embryonic stem cells, SNUhES32. Cell survival, proliferation, apoptosis, and differentiation on days 1, 3, 5, and 7 post treatment were analyzed. Treatment with 6% sevoflurane increased cell viability (P = 0.046) and decreased apoptosis (P = 0.014) on day 5, but the effect did not persist on day 7. Survival and apoptosis were not affected by 3% and 8% sevoflurane; there was no effect of proliferation at any of the tested concentrations. The differentiation of cells exposed to 6% or 8% sevoflurane decreased on day 1 (P = 0.033 and P = 0.036 for 6% and 8% sevoflurane, respectively) but was again normalized on days 3-7. Clinically relevant treatment with sevoflurane for 2 h induces no significant changes in the survival, proliferation, apoptosis, and differentiation of human neural precursor cells, although supraclinical doses of sevoflurane do alter human neurogenesis transiently.

  13. Transplantation of Photoreceptor Precursors Isolated via a Cell Surface Biomarker Panel From Embryonic Stem Cell‐Derived Self‐Forming Retina

    PubMed Central

    Gonzalez‐Cordero, Anai; West, Emma L.; Han, Ya‐Ting; Welby, Emily; Naeem, Arifa; Blackford, Samuel J. I.; Bainbridge, James W. B.; Pearson, Rachael A.; Ali, Robin R.

    2015-01-01

    Abstract Loss of photoreceptors due to retinal degeneration is a major cause of untreatable blindness. Cell replacement therapy, using pluripotent stem cell‐derived photoreceptor cells, may be a feasible future treatment. Achieving safe and effective cell replacement is critically dependent on the stringent selection and purification of optimal cells for transplantation. Previously, we demonstrated effective transplantation of post‐mitotic photoreceptor precursor cells labelled by fluorescent reporter genes. As genetically labelled cells are not desirable for therapy, here we developed a surface biomarker cell selection strategy for application to complex pluripotent stem cell differentiation cultures. We show that a five cell surface biomarker panel CD73(+)CD24(+)CD133(+)CD47(+)CD15(−) facilitates the isolation of photoreceptor precursors from three‐dimensional self‐forming retina differentiated from mouse embryonic stem cells. Importantly, stem cell‐derived cells isolated using the biomarker panel successfully integrate and mature into new rod photoreceptors in the adult mouse retinae after subretinal transplantation. Conversely, unsorted or negatively selected cells do not give rise to newly integrated rods after transplantation. The biomarker panel also removes detrimental proliferating cells prior to transplantation. Notably, we demonstrate how expression of the biomarker panel is conserved in the human retina and propose that a similar selection strategy will facilitate isolation of human transplantation‐competent cells for therapeutic application. Stem Cells 2015;33:2469—2482 PMID:25982268

  14. Sensory recovery after cell therapy in peripheral nerve repair: effects of naïve and skin precursor-derived Schwann cells.

    PubMed

    Shakhbazau, Antos; Mohanty, Chandan; Kumar, Ranjan; Midha, Rajiv

    2014-08-01

    Cell therapy is a promising candidate among biological or technological innovations sought to augment microsurgical techniques in peripheral nerve repair. This report describes long-term functional regenerative effects of cell therapy in the rat injury model with a focus on sensory recovery. Schwann cells were derived from isogenic nerve or skin precursor cells and injected into the transected and immediately repaired sciatic nerve distal to the injury site. Sensory recovery was assessed at weeks 4, 7, and 10. Axonal regeneration was assessed at Week 11. By Week 10, thermal sensitivity in cell therapy groups returned to a level indistinguishable from the baseline (p > 0.05). Immunohistochemistry at 11 weeks after injury showed improved regeneration of NF+ and IB4+ axons. The results of this study show that cell therapy significantly improves thermal sensation and the number of regenerated sensory neurons at 11 weeks after injury. These findings contribute to the view of skin-derived stem cells as a reliable source of Schwann cells with therapeutic potential for functional recovery in damaged peripheral nerve.

  15. Large-scale expansion of human skin-derived precursor cells (hSKPs) in stirred suspension bioreactors.

    PubMed

    Surrao, Denver C; Boon, Kathryn; Borys, Breanna; Sinha, Sarthak; Kumar, Ranjan; Biernaskie, Jeff; Kallos, Michael S

    2016-12-01

    Human skin-derived precursor cells (hSKPs) are multipotent adult stem cells found in the dermis of human skin. Incorporation of hSKPs into split-thickness skin grafts (STSGs), the current gold standard to treat severe burns or tissue resections, has been proposed as a treatment option to enhance skin wound healing and tissue function. For this approach to be clinically viable substantial quantities of hSKPs are required, which is the rate-limiting step, as only a few thousand hSKPs can be isolated from an autologous skin biopsy without causing donor site morbidity. In order to produce sufficient quantities of clinically viable cells, we have developed a bioprocess capable of expanding hSKPs as aggregates in stirred suspension bioreactors (SSBs). In this study, we found hSKPs from adult donors to expand significantly more (P < 0.05) at 60 rpm in SSBs than in static cultures. Furthermore, the utility of the SSBs, at 60 rpm is demonstrated by serial passaging of hSKPs from a small starting population, which can be isolated from an autologous skin biopsy without causing donor site morbidity. At 60 rpm, aggregates were markedly smaller and did not experience oxygen diffusional limitations, as seen in hSKPs cultured at 40 rpm. While hSKPs also grew at 80 rpm (0.74 Pa) and 100 rpm (1 Pa), they produced smaller aggregates due to high shear stress. The pH of the media in all the SSBs was closer to biological conditions and significantly different (P < 0.05) from static cultures, which recorded acidic pH conditions. The nutrient concentrations of the media in all the SSBs and static cultures did not drop below acceptable limits. Furthermore, there was no significant build-up of waste products to limit hSKP expansion in the SSBs. In addition, hSKP markers were maintained in the 60 rpm SSB as demonstrated by immunocytochemistry. This method of growing hSKPs in a batch culture at 60 rpm in a SSB represents an important first step in developing an

  16. Estradiol promotes proliferation of dopaminergic precursors resulting in a higher proportion of dopamine neurons derived from mouse embryonic stem cells.

    PubMed

    Díaz, Néstor F; Díaz-Martínez, Néstor E; Camacho-Arroyo, Ignacio; Velasco, Iván

    2009-08-01

    Estradiol protects dopamine neurons of the substantia nigra from toxic insults. Such neurons succumb in Parkinson's disease; one strategy for restoring dopamine deficiency is cell therapy with neurons differentiated from embryonic stem cells. We investigated the effects of 17beta-estradiol on dopaminergic induction of embryonic stem cells using the 5-stage protocol. Cells were incubated with different steroid concentrations during the proliferation (stage 4) or differentiation (stage 5) phases. Estradiol added at nM concentrations only during stage 4 increases the proliferation of dopaminergic precursors expressing Lmx1a, inducing a higher proportion of dopamine neurons at stage 5. These actions were mediated by activation of estrogen receptors, because co-incubation of cells with estradiol and ICI 182,780 completely abolished the positive effect on both proliferation of committed precursors, and subsequent differentiation to dopaminergic neurons. Our results suggest that estradiol should be useful in producing higher proportions of dopamine neurons from embryonic stem cells aimed for treating Parkinson's disease.

  17. Estradiol promotes proliferation of dopaminergic precursors resulting in a higher proportion of dopamine neurons derived from mouse embryonic stem cells

    PubMed Central

    Díaz, Néstor F.; Díaz-Martínez, Néstor E.; Camacho-Arroyo, Ignacio; Velasco, Iván

    2009-01-01

    Estradiol protects dopamine neurons of the substantia nigra from toxic insults. Such neurons succumb in Parkinson’s disease; one strategy for restoring dopamine deficiency is cell therapy with neurons differentiated from embryonic stem cells. We investigated the effects of 17β-estradiol on dopaminergic induction of embryonic stem cells using the 5-stage protocol. Cells were incubated with different steroid concentrations during the proliferation (stage 4) or differentiation (stage 5) phases. Estradiol added at nM concentrations only during stage 4 increases the proliferation of dopaminergic precursors expressing Lmx1a, inducing a higher proportion of dopamine neurons at stage 5. These actions were mediated by activation of estrogen receptors, because co-incubation of cells with estradiol and ICI 182,780 completely abolished the positive effect on both proliferation of committed precursors, and subsequent differentiation to dopaminergic neurons. Our results suggest that estradiol should be useful to produce higher proportions of dopamine neurons from embryonic stem cells aimed for treating Parkinson’s disease. PMID:19379802

  18. Patient-specific iPSC-derived photoreceptor precursor cells as a means to investigate retinitis pigmentosa

    PubMed Central

    Tucker, Budd A; Mullins, Robert F; Streb, Luan M; Anfinson, Kristin; Eyestone, Mari E; Kaalberg, Emily; Riker, Megan J; Drack, Arlene V; Braun, Terry A; Stone, Edwin M

    2013-01-01

    Next-generation and Sanger sequencing were combined to identify disease-causing USH2A mutations in an adult patient with autosomal recessive RP. Induced pluripotent stem cells (iPSCs), generated from the patient’s keratinocytes, were differentiated into multi-layer eyecup-like structures with features of human retinal precursor cells. The inner layer of the eyecups contained photoreceptor precursor cells that expressed photoreceptor markers and exhibited axonemes and basal bodies characteristic of outer segments. Analysis of the USH2A transcripts of these cells revealed that one of the patient’s mutations causes exonification of intron 40, a translation frameshift and a premature stop codon. Western blotting revealed upregulation of GRP78 and GRP94, suggesting that the patient’s other USH2A variant (Arg4192His) causes disease through protein misfolding and ER stress. Transplantation into 4-day-old immunodeficient Crb1−/− mice resulted in the formation of morphologically and immunohistochemically recognizable photoreceptor cells, suggesting that the mutations in this patient act via post-developmental photoreceptor degeneration. DOI: http://dx.doi.org/10.7554/eLife.00824.001 PMID:23991284

  19. Maintenance and Repair of the Lung Endothelium Does Not Involve Contributions from Marrow-Derived Endothelial Precursor Cells

    PubMed Central

    Ohle, Sarah J.; Anandaiah, Asha; Fabian, Attila J.; Fine, Alan

    2012-01-01

    Lung endothelium is believed to be a quiescent tissue with the potential to exhibit rapid and effective repair after injury. Endothelial progenitor cells derived from the bone marrow have been proposed as one source of new endothelial cells that may directly contribute to pulmonary endothelial cell homeostasis and repair. Here we use bone marrow transplantation models, using purified hematopoietic stem cells (HSCs) or unfractionated whole marrow, to assess engraftment of cells in the endothelium of a variety of tissues. We find scant evidence for any contribution of bone marrow–derived cells to the pulmonary endothelium in the steady state or after recovery from hyperoxia-induced endothelial injury. Although a rare population of CD45−/CD31+/VECadherin+ bone marrow–derived cells, originating from HSCs, can be found in lung tissue after transplantation, these cells are not readily found in anatomic locations that define the pulmonary endothelium. Moreover, by tracking transplanted bone marrow cells obtained from donor transgenic mice containing endothelial lineage–selective reporters (Tie2-GFP), no contribution of bone marrow–derived cells to the adult lung, liver, pancreas, heart, and kidney endothelium can be detected, even after prolonged follow-up periods of 11 months or after recovery from hyperoxic pulmonary endothelial injury. Our findings argue against any significant engraftment of bone marrow–derived cells in the pulmonary vascular endothelium. PMID:22323363

  20. Remyelination Is Correlated with Regulatory T Cell Induction Following Human Embryoid Body-Derived Neural Precursor Cell Transplantation in a Viral Model of Multiple Sclerosis.

    PubMed

    Plaisted, Warren C; Zavala, Angel; Hingco, Edna; Tran, Ha; Coleman, Ronald; Lane, Thomas E; Loring, Jeanne F; Walsh, Craig M

    2016-01-01

    We have recently described sustained clinical recovery associated with dampened neuroinflammation and remyelination following transplantation of neural precursor cells (NPCs) derived from human embryonic stem cells (hESCs) in a viral model of the human demyelinating disease multiple sclerosis. The hNPCs used in that study were derived by a novel direct differentiation method (direct differentiation, DD-NPCs) that resulted in a unique gene expression pattern when compared to hNPCs derived by conventional methods. Since the therapeutic potential of human NPCs may differ greatly depending on the method of derivation and culture, we wanted to determine whether NPCs differentiated using conventional methods would be similarly effective in improving clinical outcome under neuroinflammatory demyelinating conditions. For the current study, we utilized hNPCs differentiated from a human induced pluripotent cell line via an embryoid body intermediate stage (EB-NPCs). Intraspinal transplantation of EB-NPCs into mice infected with the neurotropic JHM strain of mouse hepatitis virus (JHMV) resulted in decreased accumulation of CD4+ T cells in the central nervous system that was concomitant with reduced demyelination at the site of injection. Dampened neuroinflammation and remyelination was correlated with a transient increase in CD4+FOXP3+ regulatory T cells (Tregs) concentrated within the peripheral lymphatics. However, compared to our earlier study, pathological improvements were modest and did not result in significant clinical recovery. We conclude that the genetic signature of NPCs is critical to their effectiveness in this model of viral-induced neurologic disease. These comparisons will be useful for understanding what factors are critical for the sustained clinical improvement.

  1. Remyelination Is Correlated with Regulatory T Cell Induction Following Human Embryoid Body-Derived Neural Precursor Cell Transplantation in a Viral Model of Multiple Sclerosis

    PubMed Central

    Plaisted, Warren C.; Zavala, Angel; Hingco, Edna; Tran, Ha; Coleman, Ronald; Lane, Thomas E.; Loring, Jeanne F.; Walsh, Craig M.

    2016-01-01

    We have recently described sustained clinical recovery associated with dampened neuroinflammation and remyelination following transplantation of neural precursor cells (NPCs) derived from human embryonic stem cells (hESCs) in a viral model of the human demyelinating disease multiple sclerosis. The hNPCs used in that study were derived by a novel direct differentiation method (direct differentiation, DD-NPCs) that resulted in a unique gene expression pattern when compared to hNPCs derived by conventional methods. Since the therapeutic potential of human NPCs may differ greatly depending on the method of derivation and culture, we wanted to determine whether NPCs differentiated using conventional methods would be similarly effective in improving clinical outcome under neuroinflammatory demyelinating conditions. For the current study, we utilized hNPCs differentiated from a human induced pluripotent cell line via an embryoid body intermediate stage (EB-NPCs). Intraspinal transplantation of EB-NPCs into mice infected with the neurotropic JHM strain of mouse hepatitis virus (JHMV) resulted in decreased accumulation of CD4+ T cells in the central nervous system that was concomitant with reduced demyelination at the site of injection. Dampened neuroinflammation and remyelination was correlated with a transient increase in CD4+FOXP3+ regulatory T cells (Tregs) concentrated within the peripheral lymphatics. However, compared to our earlier study, pathological improvements were modest and did not result in significant clinical recovery. We conclude that the genetic signature of NPCs is critical to their effectiveness in this model of viral-induced neurologic disease. These comparisons will be useful for understanding what factors are critical for the sustained clinical improvement. PMID:27310015

  2. IL-23 drives differentiation of peripheral γδ17 T cells from adult bone marrow-derived precursors.

    PubMed

    Papotto, Pedro H; Gonçalves-Sousa, Natacha; Schmolka, Nina; Iseppon, Andrea; Mensurado, Sofia; Stockinger, Brigitta; Ribot, Julie C; Silva-Santos, Bruno

    2017-08-30

    Pro-inflammatory interleukin (IL)-17-producing γδ (γδ17) T cells are thought to develop exclusively in the thymus during fetal/perinatal life, as adult bone marrow precursors fail to generate γδ17 T cells under homeostatic conditions. Here, we employ a model of experimental autoimmune encephalomyelitis (EAE) in which hematopoiesis is reset by bone marrow transplantation and demonstrate unequivocally that Vγ4(+) γδ17 T cells can develop de novo in draining lymph nodes in response to innate stimuli. In vitro, γδ T cells from IL-17 fate-mapping reporter mice that had never activated the Il17 locus acquire IL-17 expression upon stimulation with IL-1β and IL-23. Furthermore, IL-23R (but not IL-1R1) deficiency severely compromises the induction of γδ17 T cells in EAE, demonstrating the key role of IL-23 in the process. Finally, we show, in a composite model involving transfers of both adult bone marrow and neonatal thymocytes, that induced γδ17 T cells make up a substantial fraction of the total IL-17-producing Vγ4(+) T-cell pool upon inflammation, which attests the relevance of this novel pathway of peripheral γδ17 T-cell differentiation. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

  3. Basic fibroblast growth factor (bFGF) acts intracellularly to cause the transdifferentiation of avian neural crest-derived Schwann cell precursors into melanocytes.

    PubMed

    Sherman, L; Stocker, K M; Morrison, R; Ciment, G

    1993-08-01

    We previously found that cultured neural crest-derived cells from embryonic quail peripheral nerves, which consist mostly of Schwann cell precursors, gave rise to melanocytes following treatment with basic fibroblast growth factor (bFGF) or 12-O-tetradecanoyl phorbol-13-acetate (TPA). Here, we show that antisense deoxyoligonucleotides targeted against two regions of the bFGF mRNA transcript blocked this TPA-induced transdifferentiation of Schwann cell precursors. Neither sense nor scrambled antisense control oligonucleotides had any effect in this regard. TPA increased bFGF protein expression in cell lysates but not in conditioned media from these cultures, and this expression was localized to the nucleus and cytoplasm. Furthermore, bFGF-neutralizing antibodies and inositol-hexakisphosphate (InsP6) both inhibited pigmentation caused by exogenous bFGF, but had no affect on TPA-induced melanogenesis, suggesting that bFGF is not released by these cells. These data indicate that bFGF is necessary for the TPA-induced transdifferentiation of Schwann cell precursors into melanocytes and that bFGF acts via an intracrine mechanism.

  4. The Ether Lipid Precursor Hexadecylglycerol Stimulates the Release and Changes the Composition of Exosomes Derived from PC-3 Cells*

    PubMed Central

    Phuyal, Santosh; Skotland, Tore; Hessvik, Nina Pettersen; Simolin, Helena; Øverbye, Anders; Brech, Andreas; Parton, Robert G.; Ekroos, Kim; Sandvig, Kirsten; Llorente, Alicia

    2015-01-01

    Exosomes are vesicles released by cells after fusion of multivesicular bodies with the plasma membrane. In this study, we have investigated whether ether lipids affect the release of exosomes in PC-3 cells. To increase the cellular levels of ether lipids, the ether lipid precursor hexadecylglycerol was added to cells. Lipidomic analysis showed that this compound was in fact able to double the cellular levels of ether lipids in these cells. Furthermore, increased levels of ether lipids were also found in exosomes released by cells containing high levels of these lipids. Interestingly, as measured by nanoparticle tracking analysis, cells containing high levels of ether lipids released more exosomes than control cells, and these exosomes were similar in size to control exosomes. Moreover, silver staining and Western blot analyses showed that the protein composition of exosomes released in the presence of hexadecylglycerol was changed; the levels of some proteins were increased, and the levels of others were reduced. In conclusion, this study clearly shows that an increase in cellular ether lipids is associated with changes in the release and composition of exosomes. PMID:25519911

  5. Gallium nitride induces neuronal differentiation markers in neural stem/precursor cells derived from rat cerebral cortex.

    PubMed

    Chen, Chi-Ruei; Li, Yi-Chen; Young, Tai-Horng

    2009-09-01

    In the present study, gallium nitride (GaN) was used as a substrate to culture neural stem/precursor cells (NSPCs), isolated from embryonic rat cerebral cortex, to examine the effect of GaN on the behavior of NSPCs in the presence of basic fibroblast growth factor (bFGF) in serum-free medium. Morphological studies showed that neurospheres maintained their initial shape and formed many long and thick processes with the fasciculate feature on GaN. Immunocytochemical characterization showed that GaN could induce the differentiation of NSPCs into neurons and astrocytes. Compared to poly-d-lysine (PDL), the most common substrate used for culturing neurons, there was considerable expression of synapsin I for differentiated neurons on GaN, suggesting GaN could induce the differentiation of NSPCs towards the mature differentiated neurons. Western blot analysis showed that the suppression of glycogen synthase kinase-3beta (GSK-3beta) activity was one of the effects of GaN-promoted NSPC differentiation into neurons. Finally, compared to PDL, GaN could significantly improve cell survival to reduce cell death after long-term culture. These results suggest that GaN potentially has a combination of electric characteristics suitable for developing neuron and/or NSPC chip systems.

  6. Contralaterally transplanted human embryonic stem cell-derived neural precursor cells (ENStem-A) migrate and improve brain functions in stroke-damaged rats.

    PubMed

    Chang, Da-Jeong; Oh, Seung-Hun; Lee, Nayeon; Choi, Chunggab; Jeon, Iksoo; Kim, Hyun Sook; Shin, Dong Ah; Lee, Seo Eun; Kim, Daehong; Song, Jihwan

    2013-11-15

    The transplantation of neural precursor cells (NPCs) is known to be a promising approach to ameliorating behavioral deficits after stroke in a rodent model of middle cerebral artery occlusion (MCAo). Previous studies have shown that transplanted NPCs migrate toward the infarct region, survive and differentiate into mature neurons to some extent. However, the spatiotemporal dynamics of NPC migration following transplantation into stroke animals have yet to be elucidated. In this study, we investigated the fates of human embryonic stem cell (hESC)-derived NPCs (ENStem-A) for 8 weeks following transplantation into the side contralateral to the infarct region using 7.0T animal magnetic resonance imaging (MRI). T2- and T2*-weighted MRI analyses indicated that the migrating cells were clearly detectable at the infarct boundary zone by 1 week, and the intensity of the MRI signals robustly increased within 4 weeks after transplantation. Afterwards, the signals were slightly increased or unchanged. At 8 weeks, we performed Prussian blue staining and immunohistochemical staining using human-specific markers, and found that high percentages of transplanted cells migrated to the infarct boundary. Most of these cells were CXCR4-positive. We also observed that the migrating cells expressed markers for various stages of neural differentiation, including Nestin, Tuj1, NeuN, TH, DARPP-32 and SV38, indicating that the transplanted cells may partially contribute to the reconstruction of the damaged neural tissues after stroke. Interestingly, we found that the extent of gliosis (glial fibrillary acidic protein-positive cells) and apoptosis (TUNEL-positive cells) were significantly decreased in the cell-transplanted group, suggesting that hESC-NPCs have a positive role in reducing glia scar formation and cell death after stroke. No tumors formed in our study. We also performed various behavioral tests, including rotarod, stepping and modified neurological severity score tests, and

  7. Delayed transplantation of precursor cell-derived astrocytes provides multiple benefits in a rat model of Parkinsons.

    PubMed

    Proschel, Christoph; Stripay, Jennifer L; Shih, Chung-Hsuan; Munger, Joshua C; Noble, Mark D

    2014-04-01

    In addition to dopaminergic neuron loss, it is clear that Parkinson disease includes other pathological changes, including loss of additional neuronal populations. As a means of addressing multiple pathological changes with a single therapeutically-relevant approach, we employed delayed transplantation of a unique class of astrocytes, GDAs(BMP), that are generated in vitro by directed differentiation of glial precursors. GDAs(BMP) produce multiple agents of interest as treatments for PD and other neurodegenerative disorders, including BDNF, GDNF, neurturin and IGF1. GDAs(BMP) also exhibit increased levels of antioxidant pathway components, including levels of NADPH and glutathione. Delayed GDA(BMP) transplantation into the 6-hydroxydopamine lesioned rat striatum restored tyrosine hydroxylase expression and promoted behavioral recovery. GDA(BMP) transplantation also rescued pathological changes not prevented in other studies, such as the rescue of parvalbumin(+) GABAergic interneurons. Consistent with expression of the synaptic modulatory proteins thrombospondin-1 and 2 by GDAs(BMP), increased expression of the synaptic protein synaptophysin was also observed. Thus, GDAs(BMP) offer a multimodal support cell therapy that provides multiple benefits without requiring prior genetic manipulation.

  8. Delayed transplantation of precursor cell-derived astrocytes provides multiple benefits in a rat model of Parkinsons

    PubMed Central

    Proschel, Christoph; Stripay, Jennifer L; Shih, Chung-Hsuan; Munger, Joshua C; Noble, Mark D

    2014-01-01

    In addition to dopaminergic neuron loss, it is clear that Parkinson disease includes other pathological changes, including loss of additional neuronal populations. As a means of addressing multiple pathological changes with a single therapeutically-relevant approach, we employed delayed transplantation of a unique class of astrocytes, GDAsBMP, that are generated in vitro by directed differentiation of glial precursors. GDAsBMP produce multiple agents of interest as treatments for PD and other neurodegenerative disorders, including BDNF, GDNF, neurturin and IGF1. GDAsBMP also exhibit increased levels of antioxidant pathway components, including levels of NADPH and glutathione. Delayed GDABMP transplantation into the 6-hydroxydopamine lesioned rat striatum restored tyrosine hydroxylase expression and promoted behavioral recovery. GDABMP transplantation also rescued pathological changes not prevented in other studies, such as the rescue of parvalbumin+ GABAergic interneurons. Consistent with expression of the synaptic modulatory proteins thrombospondin-1 and 2 by GDAsBMP, increased expression of the synaptic protein synaptophysin was also observed. Thus, GDAsBMP offer a multimodal support cell therapy that provides multiple benefits without requiring prior genetic manipulation. PMID:24477866

  9. The Innate Lymphoid Cell Precursor.

    PubMed

    Ishizuka, Isabel E; Constantinides, Michael G; Gudjonson, Herman; Bendelac, Albert

    2016-05-20

    The discovery of tissue-resident innate lymphoid cell populations effecting different forms of type 1, 2, and 3 immunity; tissue repair; and immune regulation has transformed our understanding of mucosal immunity and allergy. The emerging complexity of these populations along with compounding issues of redundancy and plasticity raise intriguing questions about their precise lineage relationship. Here we review advances in mapping the emergence of these lineages from early lymphoid precursors. We discuss the identification of a common innate lymphoid cell precursor characterized by transient expression of the transcription factor PLZF, and the lineage relationships of innate lymphoid cells with conventional natural killer cells and lymphoid tissue inducer cells. We also review the rapidly growing understanding of the network of transcription factors that direct the development of these lineages.

  10. Efficient derivation of human cardiac precursors and cardiomyocytes from pluripotent human embryonic stem cells with small molecule induction.

    PubMed

    Parsons, Xuejun H; Teng, Yang D; Parsons, James F; Snyder, Evan Y; Smotrich, David B; Moore, Dennis A

    2011-11-03

    To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic. To overcome these obstacles, we have resolved the elements of a defined culture

  11. Mevalonate-derived isopentenyl diphosphate is the biosynthetic precursor of ubiquinone prenyl side chain in tobacco BY-2 cells.

    PubMed Central

    Disch, A; Hemmerlin, A; Bach, T J; Rohmer, M

    1998-01-01

    Study of the incorporation of 13C-labelled glucose or pyruvate into the isoprenoids of tobacco BY-2 cells allowed the biosynthetic origin of isopentenyl diphosphate to be determined. Sterols synthesized in the cytoplasm and the prenyl chain of ubiquinone Q10 located in mitochondria were derived from the same isopentenyl diphosphate pool, synthesized from acetyl-CoA through mevalonate, whereas the prenyl chain of plastoquinone was obtained from the mevalonate-independent glyceraldehyde 3-phosphate/pyruvate route, like all chloroplast isoprenoids from higher plants. These results are in accord with the compartmentation and complete enzymic independence of the biosynthesis of long-chain all-trans polyprenols in mitochondria and chloroplasts. PMID:9531505

  12. Pentosan polysulfate promotes proliferation and chondrogenic differentiation of adult human bone marrow-derived mesenchymal precursor cells

    PubMed Central

    2010-01-01

    Introduction This study was undertaken to determine whether the anti-osteoarthritis drug pentosan polysulfate (PPS) influenced mesenchymal precursor cell (MPC) proliferation and differentiation. Methods Human MPCs were maintained in monolayer, pellet or micromass cultures (MMC) for up to 10 days with PPS at concentrations of 0 to 20 μg/ml. MPC viability and proliferation was assessed using the WST-1 assay and 3H-thymidine incorporation into DNA, while apoptosis was monitored by flow cytometry. Proteoglycan (PG) biosynthesis was determined by 35SO42- incorporation and staining with Alcian blue. Proteoglycan and collagen type I and collagen type II deposition in pellet cultures was also examined by Toluidine blue and immunohistochemical staining, respectively. The production of hyaluronan (HA) by MPCs in MMC was assessed by ELISA. The relative outcome of PPS, HA, heparin or dextran sulfate (DS) on PG synthesis was compared in 5-day MMC. Gene expression of MPCs in 7-day and 10-day MMC was examined using real-time PCR. MPC differentiation was investigated by co-culturing with PPS in osteogenic or adipogenic inductive culture media for 28 days. Results Significant MPC proliferation was evident by day 3 at PPS concentrations of 1 to 5 μg/ml (P < 0.01). In the presence of 1 to 10 μg/ml PPS, a 38% reduction in IL-4/IFNγ-induced MPC apoptosis was observed. In 5-day MMC, 130% stimulation of PG synthesis occurred at 2.5 μg/ml PPS (P < 0.0001), while 5.0 μg/ml PPS achieved maximal stimulation in the 7-day and 10-day cultures (P < 0.05). HA and DS at ≥ 5 μg/ml inhibited PG synthesis (P < 0.05) in 5-day MMC. Collagen type II deposition by MMC was significant at ≥ 0.5 μg/ml PPS (P < 0.001 to 0.05). In MPC-PPS pellet cultures, more PG, collagen type II but less collagen type I was deposited than in controls. Real-time PCR results were consistent with the protein data. At 5 and 10 μg/ml PPS, MPC osteogenic differentiation was suppressed (P < 0.01). Conclusions This is

  13. Insulin-like growth factor-I is a differentiation factor for postmitotic CNS stem cell-derived neuronal precursors: distinct actions from those of brain-derived neurotrophic factor.

    PubMed

    Arsenijevic, Y; Weiss, S

    1998-03-15

    Insulin-like growth factor-I (IGF-I) has been reported previously to promote the proliferation, survival, and maturation of sympathetic neuroblasts, the genesis of retinal neurons, and the survival of CNS projection and motor neurons. Here we asked whether IGF-I could promote the in vitro differentiation of postmitotic mammalian CNS neuronal precursors derived from multipotent epidermal growth factor (EGF)-responsive stem cells. In the absence of IGF-I, virtually no neurons were present in cultured stem cell progeny, whereas IGF-I increased neuron number by eight- to 40-fold. Brief exposures (2 hr) to IGF-I were sufficient to allow for neuronal differentiation without affecting proliferation or survival. IGF-I actions could be mimicked by insulin and IGF-II at concentrations that correspond to the pharmacology of the IGF-I receptor, the latter for which the mRNA was detected in undifferentiated stem cell progeny. Although ineffectual alone at low concentrations (10 nM) that would activate its own receptor, insulin was able to potentiate the actions of IGF-I by acting on mitotically active neural precursors. When neuronal precursor differentiation by IGF-I was examined in relation to brain-derived neurotrophic factor (BDNF), two important observations were made: (1) BDNF could potentiate the differentiating actions of IGF-I plus insulin, and (2) BDNF could act on a separate population of precursors that did not require IGF-I plus insulin for differentiation. Taken together, these results suggest that IGF-I and BDNF may act together or sequentially to promote neuronal precursor differentiation.

  14. Neural Precursors Derived from Embryonic Stem Cells, but Not Those from Fetal Ventral Mesencephalon, Maintain the Potential to Differentiate into Dopaminergic Neurons After Expansion In Vitro

    PubMed Central

    Chung, Sangmi; Shin, Byoung-Soo; Hwang, Michelle; Lardaro, Thomas; Kang, Un Jung; Isacson, Ole; Kima, Kwang-Soo

    2008-01-01

    Neural precursors (NPs) derived from ventral mesencephalon (VM) normally generate dopaminergic (DA) neurons in vivo but lose their potential to differentiate into DA neurons during mitogenic expansion in vitro, hampering their efficient use as a transplantable and experimental cell source. Because embryonic stem (ES) cell-derived NPs (ES NP) do not go through the same maturation process during in vitro expansion, we hypothesized that expanded ES NPs may maintain their potential to differentiate into DA neurons. To address this, we expanded NPs derived from mouse embryonic day-12.5 (E12.5) VM or ES cells and compared their developmental properties. Interestingly, expanded ES NPs fully sustain their ability to differentiate to the neuronal as well as to the DA fate. In sharp contrast, VM NPs almost completely lost their ability to become neurons and tyrosine hydroxylase-positive (TH+) neurons after expansion. Expanded ES NP-derived TH+ neurons coexpressed additional DA markers such as dopa decarboxylase and DAT (dopamine transporter). Furthermore, they also expressed other midbrain DA markers, including Nurr1 and Pitx3, and released significant amounts of DA. We also found that these ES NPs can be cryopreserved without losing their proliferative and developmental potential. Finally, we tested the in vivo characteristics of the expanded NPs derived from J1 ES cells with low passage number. When transplanted into the mouse striatum, the expanded NPs as well as control NPs efficiently generated DA neurons expressing mature DA markers, with approximately 10% tumor formation in both cases. We conclude that ES NPs maintain their developmental potential during in vitro expansion, whereas mouse E12.5 VM NPs do not. PMID:16543488

  15. Tryptase precursors are preferentially and spontaneously released, whereas mature tryptase is retained by HMC-1 cells, Mono-Mac-6 cells, and human skin-derived mast cells.

    PubMed

    Schwartz, Lawrence B; Min, Hae-Ki; Ren, Shunlin; Xia, Han-Zhang; Hu, Jiang; Zhao, Wei; Moxley, George; Fukuoka, Yoshihiro

    2003-06-01

    Tryptase (alpha and beta) levels in serum are used to assess mast cell involvement in human disease. Using cultured cells, the current study examines the hypothesis that protryptase(s) are spontaneously secreted by mast cells at rest, whereas mature tryptase(s) are stored in secretory granules until their release by activated cells. HMC-1 cells have only beta-tryptase genes and the corresponding mRNA. Mono-Mac-6 cells have both alpha- and beta-tryptase genes but preferentially express alpha-tryptase. Mono-Mac-6 cells spontaneously secrete most of their tryptase, which consists of alpha-protryptase, whereas mature tryptase is retained inside these cells. HMC-1 cells also spontaneously secrete most of their tryptase, identified as beta-protryptase, and retain mature tryptase. Skin-derived mast cells retain most of their tryptase, which is mature, and spontaneously secrete protryptase(s). Total tryptase levels in plasma are detectable but no different in healthy subjects with and without the gene for alpha-tryptase, consistent with pro forms of both alpha- and beta-tryptase being spontaneously secreted. Thus, protryptase(s) are spontaneously secreted by resting mast cells, whereas mature tryptase is retained by mast cells until they are activated to degranulate.

  16. Baccatin III, a precursor for the semisynthesis of paclitaxel, inhibits the accumulation and suppressive activity of myeloid-derived suppressor cells in tumor-bearing mice.

    PubMed

    Lee, Young-Hee; Lee, Young-Ran; Park, Chan-Su; Im, Sun-A; Song, Sukgil; Hong, Jin Tae; Whang, Bang Yeon; Kim, Kyungjae; Lee, Chong-Kil

    2014-08-01

    Myeloid-derived suppressor cells (MDSCs) mediate tumor-associated immune suppression in both cancer patients and tumor-bearing animals. Reduction or elimination of MDSCs reduces the rate of tumor progression and improves cancer therapies that employ mechanisms of immunity. Here we show that baccatin III, which is the precursor for the semisynthesis of paclitaxel, exerts anti-tumor immunomodulatory activity in very low doses (0.05-0.5mg/kg), although it is regarded as an inactive derivative of paclitaxel. Oral administration of baccatin III significantly reduced the growth of tumors induced by engrafting BALB/c mice with either 4 T1 mammary carcinoma or CT26 colon cancer cells. Baccatin III (0.5mg/kg) did not exert anti-tumor activity in athymic nude mice. Baccatin III decreased the accumulation of MDSCs in the spleens of the tumor-bearing mice. Furthermore, MDSCs isolated from baccatin III-treated mice, compared with those isolated from vehicle-treated mice, had a significantly reduced suppressive effect on T cells treated with the anti-CD3 and anti-CD28 monoclonal antibodies. Moreover, these cells produced significantly reduced amounts of reactive oxygen species and nitric oxide. These results suggest that baccatin III reduced tumor progression by inhibiting the accumulation and suppressive function of MDSCs. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Skin-derived neural precursors competitively generate functional myelin in adult demyelinated mice.

    PubMed

    Mozafari, Sabah; Laterza, Cecilia; Roussel, Delphine; Bachelin, Corinne; Marteyn, Antoine; Deboux, Cyrille; Martino, Gianvito; Baron-Van Evercooren, Anne

    2015-09-01

    Induced pluripotent stem cell-derived (iPS-derived) neural precursor cells may represent the ideal autologous cell source for cell-based therapy to promote remyelination and neuroprotection in myelin diseases. So far, the therapeutic potential of reprogrammed cells has been evaluated in neonatal demyelinating models. However, the repair efficacy and safety of these cells has not been well addressed in the demyelinated adult CNS, which has decreased cell plasticity and scarring. Moreover, it is not clear if these induced pluripotent-derived cells have the same reparative capacity as physiologically committed CNS-derived precursors. Here, we performed a side-by-side comparison of CNS-derived and skin-derived neural precursors in culture and following engraftment in murine models of adult spinal cord demyelination. Grafted induced neural precursors exhibited a high capacity for survival, safe integration, migration, and timely differentiation into mature bona fide oligodendrocytes. Moreover, grafted skin-derived neural precursors generated compact myelin around host axons and restored nodes of Ranvier and conduction velocity as efficiently as CNS-derived precursors while outcompeting endogenous cells. Together, these results provide important insights into the biology of reprogrammed cells in adult demyelinating conditions and support use of these cells for regenerative biomedicine of myelin diseases that affect the adult CNS.

  18. Chemo-sensitivity in a panel of B-cell precursor acute lymphoblastic leukemia cell lines, YCUB series, derived from children.

    PubMed

    Goto, Hiroaki; Naruto, Takuya; Tanoshima, Reo; Kato, Hiromi; Yokosuka, Tomoko; Yanagimachi, Masakatsu; Fujii, Hisaki; Yokota, Shumpei; Komine, Hiromi

    2009-10-01

    Sensitivity to 10 anticancer drugs was evaluated in 6 childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cell lines. Authenticity of newly established cell lines was confirmed by genomic fingerprinting. The line YCUB-5R established at relapse was more resistant to 4-hydroperoxy-cyclophosphamide, cytarabine, L-asparaginase, topotecan, fludarabine, and etoposide than YCUB-5 from the same patient at diagnosis. Of the drugs tested, etoposide and SN-38 (irinotecan) showed highest efficacy in the panel, with 50% growth inhibition at 0.22-1.8 microg/ml and 0.57-3.6 ng/ml, respectively. This cell line panel offers an in vitro model for the development of new therapies for childhood BCP-ALL.

  19. Proteome analysis of post-transplantation recovery mechanisms of an EAE model of multiple sclerosis treated with embryonic stem cell-derived neural precursors.

    PubMed

    Fazeli, Abolhassan Shahzadeh; Nasrabadi, Davood; Pouya, Alireza; Mirshavaladi, Shahaboodin; Sanati, Mohammad Hossein; Baharvand, Hossein; Salekdeh, Ghasem Hosseini

    2013-12-06

    Multiple sclerosis (MS) is a chronic inflammatory and progressive disorder of the central nervous system (CNS), which ultimately causes demyelination and subsequent axonal injury. Experimental autoimmune encephalomyelitis (EAE) is a well-characterized animal model to study the etiology and pathogenesis of MS. This model can also be used to investigate various therapeutic approaches for MS. Herein; we have treated a score 3 EAE mouse model with an embryonic stem cell-derived neural precursor. Clinical analysis showed recovery of the EAE model of MS following transplantation. We analyzed the proteome of spinal cords of healthy and EAE samples before and after transplantation. Proteome analysis revealed that expressions of 86 spinal cord protein spots changed in the EAE or transplanted mouse compared to controls. Mass spectrometry resulted in identification of 72 proteins. Of these, the amounts of 27 differentially expressed proteins in EAE samples returned to sham levels after transplantation, suggesting a possible correlation between changes at the proteome level and clinical signs of EAE in transplanted mice. The recovered proteins belonged to various functional groups that included disturbances in ionic and neurotransmitter release, apoptosis, iron hemostasis, and signal transduction. Our results provided a proteomic view of the molecular mechanisms of EAE recovery after stem cell transplantation. In this study, we applied proteomics to analyze the changes in proteome pattern of EAE mouse model after embryonic stem cell-derived neural precursor transplantation. Our proteome results clearly showed that the expression levels of several differentially expressed proteins in EAE samples returned to sham levels after transplantation, which suggested a possible correlation between changes at the proteome level and decreased clinical signs of EAE in transplanted mice. These results will serve as a basis to address new questions and design new experiments to elucidate the

  20. Dysfunction of Platelet-derived Growth Factor Receptor α (PDGFRα) Represses the Production of Oligodendrocytes from Arylsulfatase A-deficient Multipotential Neural Precursor Cells*

    PubMed Central

    Pituch, Katarzyna C.; Moyano, Ana L.; Lopez-Rosas, Aurora; Marottoli, Felecia M.; Li, Guannan; Hu, Chenqi; van Breemen, Richard; Månsson, Jan E.; Givogri, Maria I.

    2015-01-01

    The membrane-bound receptor for platelet-derived growth factor A (PDGFRα) is crucial for controlling the production of oligodendrocytes (OLs) for myelination, but regulation of its activity during OL differentiation is largely unknown. We have examined the effect of increased sulfated content of galactosylceramides (sulfatides) on the regulation of PDGFRα in multipotential neural precursors (NPs) that are deficient in arylsulfatase A (ASA) activity. This enzyme is responsible for the lysosomal hydrolysis of sulfatides. We show that sulfatide accumulation significantly impacts the formation of OLs via deregulation of PDGFRα function. PDGFRα is less associated with detergent-resistant membranes in ASA-deficient cells and showed a significant decrease in AKT phosphorylation. Rescue experiments with ASA showed a normalization of the ratio of long versus short sulfatides, restored PDGFRα levels, corrected its localization to detergent-resistant membranes, increased AKT phosphorylation, and normalized the production of OLs in ASA-deficient NPs. Moreover, our studies identified a novel mechanism that regulates the secretion of PDGFRα in NPs, in glial cells, and in the brain cortex via exosomal shedding. Our study provides a first step in understanding the role of sulfatides in regulating PDGFRα levels in OLs and its impact in myelination. PMID:25605750

  1. Synthesis and antiprotozoal activity of original porphyrin precursors and derivatives.

    PubMed

    Abada, Zahra; Cojean, Sandrine; Pomel, Sébastien; Ferrié, Laurent; Akagah, Bernardin; Lormier, Anh Tuan; Loiseau, Philippe M; Figadère, Bruno

    2013-09-01

    Importance of heme in African trypanosomes, Leishmania sp. and Plasmodium sp. metabolisms justifies considering the potential of porphyrins and their precursors and derivatives as potential antiparasitic agents by interfering with heme metabolism. Consequently, twenty-four porphyrin precursors and derivatives were evaluated against Leishmania donovani, Trypanosoma brucei and Plasmodium sp. The best active compound against Trypanosoma brucei brucei was a new porphyrin derivative; compound 4i, with a MEC value of 6.25 μM justifying further in vivo evaluation. Whereas these compounds were not active against intramacrophage amastigotes of L. donovani, another new porphyrin derivative, compound 4f was active in vitro against Plasmodium falciparum at 20 nM and a slight delay of mice survival was observed on the Plasmodium berghei/Swiss mice model at 50 μmol/kg/day × 4. Pharmacomodulations should be further developed relying on a better knowledge on the porphyrin behaviour into the parasites comparatively to host cells.

  2. Phenotypic and functional activation of hyporesponsive KIRnegNKG2Aneg human NK-cell precursors requires IL12p70 provided by Poly(I:C)-matured monocyte-derived dendritic cells.

    PubMed

    Curran, Shane A; Romano, Emanuela; Kennedy, Michael G; Hsu, Katharine C; Young, James W

    2014-10-01

    A functionally responsive natural killer (NK)-cell repertoire requires the acquisition of inhibitory NKG2A and killer immunoglobulin-like receptors (KIR) through pathways that remain undefined. Functional donor NK cells expressing KIRs for non-self class I MHC ligands contribute to a positive outcome after allogeneic hematopoietic stem cell transplantation (alloHSCT) by targeting HLA-matched recipient leukemic cells. Insofar as circulating donor conventional dendritic cells (DC) reconstitute with comparable kinetics with donor NK cells after alloHSCT, we used hyporesponsive KIRnegNKG2Aneg precursor cells to evaluate how specific DC subtypes generate a functionally active NK-cell repertoire. Both monocyte-derived DCs (moDC) and Langerhans-type DCs (LC) induce KIRnegNKG2Aneg precursor cells to express the inhibitory receptors NKG2A and KIR, without requiring cell proliferation. Poly(I:C)-matured moDCs significantly augmented the expression of NKG2A, but not KIR, in an IL12p70-dependent manner. Although all DC-stimulated KIRnegNKG2Aneg cells were able to acquire cytolytic activity against class I MHC-negative targets, the ability to secrete IFNγ was restricted to cells that were stimulated by IL12p70-producing, poly(I:C)-matured moDCs. This critical ability of poly(I:C)-matured moDCs to provide IL12p70 to developing KIRnegNKG2Aneg precursors results in a dom4inant, multifunctional, NKG2Apos NK-cell population that is capable of both cytolysis and IFNγ production. Poly(I:C)-matured moDCs are, therefore, the most effective conventional DC subtype for generating a functionally competent NK-cell repertoire by an IL12p70-dependent mechanism.

  3. Novel Use of All-trans-Retinoic Acid in a Model of Lipopolysaccharide-immunosuppression to Decrease the Generation of Myeloid-derived Suppressor Cells by Reducing the Proliferation of CD34+ Precursor Cells.

    PubMed

    Martire-Greco, Daiana; Rodriguez-Rodrigues, Nahuel; Castillo, Luis A; Vecchione, María Belén; de Campos-Nebel, Marcelo; Moreno, Marlina Córdoba; Meiss, Roberto; Vermeulen, Mónica; Landoni, Veronica I; Fernandez, Gabriela C

    2016-11-25

    All-trans-Retinoic Acid (ATRA) is a derivative of vitamin A with anti-proliferative properties. Endotoxin shock and subsequent immunosuppression (IS) by lipopolysaccharide (LPS) stimulates myelopoiesis with expansion of myeloid-derived suppressor cells (MDSC). Since we have previously shown that ATRA reverses the IS state by decreasing functional MDSC, our aim was to investigate if ATRA was able to modulate MDSC generation by regulating myelopoiesis in murine hematopoietic organs. We found that ATRA administration in vivo and in vitro decreased the number of CD34+ precursor cells that were increased in IS mice. When we studied the cellular mechanisms involved, we did not find any differences in apoptosis of CD34+ precursors or in the differentiation of these cells to their mature counterparts. Surprisingly, ATRA decreased precursor proliferation, in vitro and in vivo, as assessed by a reduction in the size and number of colony forming units (CFU) generated from CD34+ cells and by a decreased incorporation of H-thymidine. Moreover, ATRA administration to IS mice decreased the number of MDSC in the spleen, with a restoration of T lymphocyte proliferation and a restitution of the histological architecture. Our results indicate, for the first time, a new use of ATRA to abolish LPS-induced myelopoiesis, affecting the proliferation of precursor cells, and in consequence, decreasing MDSC generation, having a direct impact on the improvement of immune competence. Administration of ATRA could overcome the immunosuppressive state generated by sepsis that often leads to opportunistic life-threatening infections. Therefore, ATRA could be considered a complementary treatment to enhance immune responses.

  4. Enhanced fetal hemoglobin production by phenylacetate and 4-phenylbutyrate in erythroid precursors derived from normal donors and patients with sickle cell anemia and beta-thalassemia.

    PubMed

    Fibach, E; Prasanna, P; Rodgers, G P; Samid, D

    1993-10-01

    In both sickle cell (SS) anemia and beta-thalassemia (beta-thal), an increase in fetal hemoglobin (HbF) ameliorates the clinical symptoms of the underlying disease. Several pharmacologic agents have been used to elevate HbF levels in adults; however, concerns regarding adverse effects of the prevailing drugs raise an urgent need for other agents capable of stimulating HbF production. We show here that sodium phenylacetate (NaPA) and its precursor, sodium 4-phenylbutyrate (NaPB), can enhance HbF production in cultured erythroid progenitor derived from normal donors and patients with SS anemia or beta-thal, when used at pharmacologic concentrations. Treatment resulted in (1) reduced cell proliferation, (2) elevated hemoglobin (Hb) content per cell (mean cellular Hb [MCH]), and (3) an increased proportion of HbF produced, associated with elevated levels of gamma-globin mRNA. Moreover, the active phenyl-fatty acids, with NaPA as a prototype, potentiated HbF induction by other drugs of clinical interest, including hydroxyurea (HU), sodium butyrate, and 5-azacytidine (5AzaC). Efficacy could be further enhanced by introducing chlorine substituents at the phenyl ring to increase drug lipophilicity. Our findings indicate that NaPA and NaPB, both already proven safe and effective in treatment of children with urea cycle disorders, might benefit also patients with severe hemoglobinopathies. The two-phase liquid culture procedure used in this study should prove valuable in further studies exploring the mechanisms of HbF induction by these agents, and might provide an assay to predict patient response in the clinical setting.

  5. Enzymatically degradable poly(ethylene glycol) hydrogels for the 3D culture and release of human embryonic stem cell derived pancreatic precursor cell aggregates.

    PubMed

    Amer, Luke D; Holtzinger, Audrey; Keller, Gordon; Mahoney, Melissa J; Bryant, Stephanie J

    2015-08-01

    This study aimed to develop a three dimensional culture platform for aggregates of human embryonic stem cell (hESC)-derived pancreatic progenitors that enables long-term culture, maintains aggregate size and morphology, does not adversely affect differentiation and provides a means for aggregate recovery. A platform was developed with poly(ethylene glycol) hydrogels containing collagen type I, for cell-matrix interactions, and peptide crosslinkers, for facile recovery of aggregates. The platform was first demonstrated with RIN-m5F cells, showing encapsulation and subsequent release of single cells and aggregates without adversely affecting viability. Aggregates of hESC-derived pancreatic progenitors with an effective diameter of 82 (15)μm were either encapsulated in hydrogels or cultured in suspension for 28 days. At day 14, aggregate viability was maintained in the hydrogels, but significantly reduced (88%) in suspension culture. However by day 28, viability was reduced under both culture conditions. Aggregate size was maintained in the hydrogels, but in suspension was significantly higher (∼ 2-fold) by day 28. The ability to release aggregates followed by a second enzyme treatment to achieve single cells enabled assessment by flow cytometry. Prior to encapsulation, there were 39% Pdx1(+)/Nkx6.1(+) cells, key endocrine markers required for β-cell maturation. The fraction of doubly positive cells was not affected in hydrogels but was slightly and significantly lower in suspension culture by 28 days. In conclusion, we demonstrate that a MMP-sensitive PEG hydrogel containing collagen type I is a promising platform for hESC-derived pancreatic progenitors that maintains viable aggregates, aggregate size, and progenitor state and offers facile recovery of aggregates. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  6. Transplantation of bone marrow stromal cell-derived neural precursor cells ameliorates deficits in a rat model of complete spinal cord transection.

    PubMed

    Aizawa-Kohama, Misaki; Endo, Toshiki; Kitada, Masaaki; Wakao, Shohei; Sumiyoshi, Akira; Matsuse, Dai; Kuroda, Yasumasa; Morita, Takahiro; Riera, Jorge J; Kawashima, Ryuta; Tominaga, Teiji; Dezawa, Mari

    2013-01-01

    After severe spinal cord injury, spontaneous functional recovery is limited. Numerous studies have demonstrated cell transplantation as a reliable therapeutic approach. However, it remains unknown whether grafted neuronal cells could replace lost neurons and reconstruct neuronal networks in the injured spinal cord. To address this issue, we transplanted bone marrow stromal cell-derived neural progenitor cells (BM-NPCs) in a rat model of complete spinal cord transection 9 days after the injury. BM-NPCs were induced from bone marrow stromal cells (BMSCs) by gene transfer of the Notch-1 intracellular domain followed by culturing in the neurosphere method. As reported previously, BM-NPCs differentiated into neuronal cells in a highly selective manner in vitro. We assessed hind limb movements of the animals weekly for 7 weeks to monitor functional recovery after local injection of BM-NPCs to the transected site. To test the sensory recovery, we performed functional magnetic resonance imaging (fMRI) using electrical stimulation of the hind limbs. In the injured spinal cord, transplanted BM-NPCs were confirmed to express neuronal markers 7 weeks following the transplantation. Grafted cells successfully extended neurites beyond the transected portion of the spinal cord. Adjacent localization of synaptophysin and PSD-95 in the transplanted cells suggested synaptic formations. These results indicated survival and successful differentiation of BM-NPCs in the severely injured spinal cord. Importantly, rats that received BM-NPCs demonstrated significant motor recovery when compared to the vehicle injection group. Volumes of the fMRI signals in somatosensory cortex were larger in the BM-NPC-grafted animals. However, neuronal activity was diverse and not confined to the original hind limb territory in the somatosensory cortex. Therefore, reconstruction of neuronal networks was not clearly confirmed. Our results indicated BM-NPCs as an effective method to deliver neuronal lineage

  7. Human embryonic epidermis contains a diverse Langerhans cell precursor pool.

    PubMed

    Schuster, Christopher; Mildner, Michael; Mairhofer, Mario; Bauer, Wolfgang; Fiala, Christian; Prior, Marion; Eppel, Wolfgang; Kolbus, Andrea; Tschachler, Erwin; Stingl, Georg; Elbe-Bürger, Adelheid

    2014-02-01

    Despite intense efforts, the exact phenotype of the epidermal Langerhans cell (LC) precursors during human ontogeny has not been determined yet. These elusive precursors are believed to migrate into the embryonic skin and to express primitive surface markers, including CD36, but not typical LC markers such as CD1a, CD1c and CD207. The aim of this study was to further characterize the phenotype of LC precursors in human embryonic epidermis and to compare it with that of LCs in healthy adult skin. We found that epidermal leukocytes in first trimester human skin are negative for CD34 and heterogeneous with regard to the expression of CD1c, CD14 and CD36, thus contrasting the phenotypic uniformity of epidermal LCs in adult skin. These data indicate that LC precursors colonize the developing epidermis in an undifferentiated state, where they acquire the definitive LC marker profile with time. Using a human three-dimensional full-thickness skin model to mimic in vivo LC development, we found that FACS-sorted, CD207(-) cord blood-derived haematopoietic precursor cells resembling foetal LC precursors but not CD14(+)CD16(-) blood monocytes integrate into skin equivalents, and without additional exogenous cytokines give rise to cells that morphologically and phenotypically resemble LCs. Overall, it appears that CD14(-) haematopoietic precursors possess a much higher differentiation potential than CD14(+) precursor cells.

  8. Changes in numbers and types of mast cell colony-forming cells in the peritoneal cavity of mice after injection of distilled water: evidence that mast cells suppress differentiation of bone marrow-derived precursors

    SciTech Connect

    Kanakura, Y.; Kuriu, A.; Waki, N.; Nakano, T.; Asai, H.; Yonezawa, T.; Kitamura, Y.

    1988-03-01

    Two different types of cells in the peritoneal cavity of mice produce mast cell colonies in methylcellulose. Large mast cell colonies are produced by bone marrow-derived precursors resembling lymphoid cells by light microscopy (L-CFU-Mast), whereas medium and small mast cell colonies are produced by morphologically identifiable mast cells (M-CFU-Mast and S-CFU-Mast, respectively). In the present study we eradicated peritoneal mast cells by intraperitoneal (IP) injection of distilled water. The regeneration process was investigated to clarify the relationship between L-CFU-Mast, M-CFU-Mast, and S-CFU-Mast. After injection of distilled water, M-CFU-Mast and S-CFU-Mast disappeared, but L-CFU-Mast increased, and then M-CFU-Mast and S-CFU-Mast appeared, suggesting the presence of a hierarchic relationship. When purified peritoneal mast cells were injected two days after the water injection, the L-CFU-Mast did not increase. In the peritoneal cavity of WBB6F1-+/+ mice that had been lethally irradiated and rescued by bone marrow cells of C57BL/6-bgJ/bgJ (beige, Chediak-Higashi syndrome) mice, L-CFU-Mast were of bgJ/bgJ type, but M-CFU-Mast and S-CFU-Mast were of +/+ type. The injection of distilled water to the radiation chimeras resulted in the development of bgJ/bgJ-type M-CFU-Mast and then S-CFU-Mast. The presence of mast cells appeared to suppress the recruitment of L-CFU-Mast from the bloodstream and to inhibit the differentiation of L-CFU-Mast to M-CFU-Mast.

  9. Ceramics Derived from Organo-Metallic Precursors

    DTIC Science & Technology

    1991-10-01

    rates are desired to allow the film and the substrate to adjust to the thermal expansion mismatch. An extended soaking period from 4000 to 620’C...normal alkoxide-derived coatings. Organic modification enables the refractive index, microhardness and thermal expansion coefficient of the film as...composition and thermal history in the Bi-Ca-Sr-Cu-O system. The system was selected for detailed investigation because of the attractive high temperature

  10. Rb suppresses human cone-precursor-derived retinoblastoma tumours.

    PubMed

    Xu, Xiaoliang L; Singh, Hardeep P; Wang, Lu; Qi, Dong-Lai; Poulos, Bradford K; Abramson, David H; Jhanwar, Suresh C; Cobrinik, David

    2014-10-16

    Retinoblastoma is a childhood retinal tumour that initiates in response to biallelic RB1 inactivation and loss of functional retinoblastoma (Rb) protein. Although Rb has diverse tumour-suppressor functions and is inactivated in many cancers, germline RB1 mutations predispose to retinoblastoma far more strongly than to other malignancies. This tropism suggests that retinal cell-type-specific circuitry sensitizes to Rb loss, yet the nature of the circuitry and the cell type in which it operates have been unclear. Here we show that post-mitotic human cone precursors are uniquely sensitive to Rb depletion. Rb knockdown induced cone precursor proliferation in prospectively isolated populations and in intact retina. Proliferation followed the induction of E2F-regulated genes, and depended on factors having strong expression in maturing cone precursors and crucial roles in retinoblastoma cell proliferation, including MYCN and MDM2. Proliferation of Rb-depleted cones and retinoblastoma cells also depended on the Rb-related protein p107, SKP2, and a p27 downregulation associated with cone precursor maturation. Moreover, Rb-depleted cone precursors formed tumours in orthotopic xenografts with histological features and protein expression typical of human retinoblastoma. These findings provide a compelling molecular rationale for a cone precursor origin of retinoblastoma. More generally, they demonstrate that cell-type-specific circuitry can collaborate with an initiating oncogenic mutation to enable tumorigenesis.

  11. Cryopreservation of GABAergic Neuronal Precursors for Cell-Based Therapy

    PubMed Central

    2017-01-01

    Cryopreservation protocols are essential for stem cells storage in order to apply them in the clinic. Here we describe a new standardized cryopreservation protocol for GABAergic neural precursors derived from the medial glanglionic eminence (MGE), a promising source of GABAergic neuronal progenitors for cell therapy against interneuron-related pathologies. We used 10% Me2SO as cryoprotectant and assessed the effects of cell culture amplification and cellular organization, as in toto explants, neurospheres, or individualized cells, on post-thaw cell viability and retrieval. We confirmed that in toto cryopreservation of MGE explants is an optimal preservation system to keep intact the interneuron precursor properties for cell transplantation, together with a high cell viability (>80%) and yield (>70%). Post-thaw proliferation and self-renewal of the cryopreserved precursors were tested in vitro. In addition, their migration capacity, acquisition of mature neuronal morphology, and potency to differentiate into multiple interneuron subtypes were also confirmed in vivo after transplantation. The results show that the cryopreserved precursor features remained intact and were similar to those immediately transplanted after their dissection from the MGE. We hope this protocol will facilitate the generation of biobanks to obtain a permanent and reliable source of GABAergic precursors for clinical application in cell-based therapies against interneuronopathies. PMID:28122047

  12. Novel Precursor Approached for CMC Derived by Polymer Pyrolysis

    DTIC Science & Technology

    1994-02-15

    DERIVED BY POLYMER PYROLYSIS Prepared by W. R. Schmidt FINAL TECHNICAL REPORT3 for period 15 Dec 1990 through 14 December 93 Contract F49620-91-C-0017...NOVEL PRECURSOR APPROACHES FOR CMC DERIVED F49620-91-C-0017 BY POLYMER PYROLYSIS 56. AUTHOR(S) Wayde R. Schmidt 7. PERFORMING ORGANIZATION NAME(S) AND...processing and pyrolysis . The conversion of PMVS to carbon-rich nanocrystalline silicon carbide ceramic was studied using a variety- of analytical

  13. Contribution of the 37-kDa laminin receptor precursor in the anti-metastatic PSP94-derived peptide PCK3145 cell surface binding

    SciTech Connect

    Annabi, Borhane; Currie, Jean-Christophe; Bouzeghrane, Mounia; Dulude, Helene; Daigneault, Luc; Garde, Seema; Rabbani, Shafaat A.; Panchal, Chandra; Wu, Jinzi J.; Beliveau, Richard . E-mail: oncomol@nobel.si.uqam.ca

    2006-07-21

    Purpose: PCK3145 is an anti-metastatic synthetic peptide with promising therapeutic efficacy against hormone-refractory prostate cancer. The characterization of the PCK3145 peptide cell surface binding/internalization mechanisms and of the receptors involved remained to be explored. Results: [{sup 14}C]PCK3145 cell surface binding assays showed rapid and transient kinetic profile, that was inhibited by RGD peptides, laminin, hyaluronan, and type-I collagen. RGD peptides were however unable to inhibit PCK3145 intracellular uptake. Far-Western ligand binding studies enabled the identification of the 37-kDa laminin receptor precursor (37LRP) as a potential ligand for PCK3145. Overexpression of the recombinant 37LRP indeed led to an increase in PCK3145 binding but unexpectedly not to its uptake. Conclusions: Our data support the implication of laminin receptors in cell surface binding and in transducing PCK3145 anti-metastatic effects, and provide a rational for targeting cancers that express high levels of such laminin receptors.

  14. CXCL16 recruits bone marrow-derived fibroblast precursors in renal fibrosis.

    PubMed

    Chen, Gang; Lin, Song-Chang; Chen, Jiyuan; He, Liqun; Dong, Feixia; Xu, Jing; Han, Shuhua; Du, Jie; Entman, Mark L; Wang, Yanlin

    2011-10-01

    Although fibroblasts are responsible for the production and deposition of extracellular matrix in renal fibrosis, their origin is controversial. Circulating fibroblast precursors may contribute to the pathogenesis of renal fibrosis, but the signaling mechanisms underlying the recruitment of bone marrow-derived fibroblast precursors into the kidney in response to injury are incompletely understood. Here, in the unilateral ureteral obstruction model of renal fibrosis, tubular epithelial cells upregulated the chemokine CXCL16 in obstructed kidneys, and circulating fibroblast precursors expressed the CXCL16 receptor, CXCR6. Compared with wild-type mice, CXCL16-knockout mice accumulated significantly fewer bone marrow-derived fibroblast precursors in obstructed kidneys. CXCL16-knockout mice also exhibited significantly fewer CD45-, collagen I-, and CXCR6-triple-positive fibroblast precursors in injured kidneys. Furthermore, targeted deletion of CXCL16 inhibited myofibroblast activation, reduced collagen deposition, and suppressed expression of collagen I and fibronectin. In conclusion, CXCL16 contributes to the pathogenesis of renal fibrosis by recruiting bone marrow-derived fibroblast precursors.

  15. In vitro activity of aplidine, a new marine-derived anti-cancer compound, on freshly explanted clonogenic human tumour cells and haematopoietic precursor cells.

    PubMed

    Depenbrock, H; Peter, R; Faircloth, G T; Manzanares, I; Jimeno, J; Hanauske, A R

    1998-09-01

    Aplidine is a new marine anti-cancer depsipeptide isolated from the Mediterranean tunicate Aplidium albicans. We have evaluated its antiproliferative action against a variety of freshly explanted human tumour specimens. Concentration ranges of 0.01-1.0 microM and 0.0001-1.0 microM were used in short- and long-term exposure schedules respectively. After exposure for 1 h in 49 evaluable specimens, aplidine showed a clear concentration-dependent anti-tumour effect. At 0.05 microM, 85% of the specimens were markedly inhibited. Continuous exposure for 21-28 days in 54 tumour specimens also led to a concentration-dependent activity relationship. Fifty per cent and 100% tumour inhibitions were achieved with 0.001 microM and 0.05 microM respectively. A head to head evaluation assessing short vs continuous exposure was carried out, resulting in evidence of an activity-time of exposure relationship. Breast, melanoma and non-small-cell lung cancer appear to be sensitive to low concentrations of aplidine. In addition the evaluation of the effects of aplidine on haematopoietic cells showed a concentration-dependent toxicity. However, under continuous exposure, active concentrations induced mild bone marrow toxicity, indicating that a therapeutic window at marginally myelotoxic concentrations might exist.

  16. In vitro activity of aplidine, a new marine-derived anti-cancer compound, on freshly explanted clonogenic human tumour cells and haematopoietic precursor cells.

    PubMed Central

    Depenbrock, H.; Peter, R.; Faircloth, G. T.; Manzanares, I.; Jimeno, J.; Hanauske, A. R.

    1998-01-01

    Aplidine is a new marine anti-cancer depsipeptide isolated from the Mediterranean tunicate Aplidium albicans. We have evaluated its antiproliferative action against a variety of freshly explanted human tumour specimens. Concentration ranges of 0.01-1.0 microM and 0.0001-1.0 microM were used in short- and long-term exposure schedules respectively. After exposure for 1 h in 49 evaluable specimens, aplidine showed a clear concentration-dependent anti-tumour effect. At 0.05 microM, 85% of the specimens were markedly inhibited. Continuous exposure for 21-28 days in 54 tumour specimens also led to a concentration-dependent activity relationship. Fifty per cent and 100% tumour inhibitions were achieved with 0.001 microM and 0.05 microM respectively. A head to head evaluation assessing short vs continuous exposure was carried out, resulting in evidence of an activity-time of exposure relationship. Breast, melanoma and non-small-cell lung cancer appear to be sensitive to low concentrations of aplidine. In addition the evaluation of the effects of aplidine on haematopoietic cells showed a concentration-dependent toxicity. However, under continuous exposure, active concentrations induced mild bone marrow toxicity, indicating that a therapeutic window at marginally myelotoxic concentrations might exist. PMID:9743292

  17. Ferritin H subunit gene is specifically expressed in melanophore precursor-derived white pigment cells in which reflecting platelets are formed from stage II melanosomes in the periodic albino mutant of Xenopus laevis.

    PubMed

    Fukuzawa, Toshihiko

    2015-09-01

    "White pigment cells" are derived from melanophore precursors and contain both melanophore-specific and iridophore-specific pigment organelles. Whereas melanophores differentiate in the wild type regenerating tail, white pigment cells appear in the regenerating tail in the periodic albino mutant (a(p)/a(p)) of Xenopus laevis. The localization and density of white pigment cells in the mutant regenerating tail are similar to those of melanophores in the wild type regenerating tail. Here, white pigment cells in the mutant regenerating tail have been compared with melanophores in the wild type regenerating tail in the presence of phenylthiourea (PTU), which inhibits melanosome maturation in melanophores but does not affect reflecting platelet formation in white pigment cells. Ultrastructural analysis shows that reflecting platelet formation in white pigment cells is different from that in iridophores. Reflecting platelets in iridophores are formed from spherical vesicles with electron-dense material, whereas they are formed from stage II melanosomes characteristic of melanophore precursors in white pigment cells. Ultrastructural features of pigment organelles, except reflecting platelets, are similar between mutant melanophores and white pigment cells. In an attempt to identify specific genes in white pigment cells, a subtracted cDNA library enriched for mutant cDNAs has been prepared. Subtracted cDNA fragments have been cloned and selected by whole mount in situ hybridization. Among cDNA fragments examined so far, the ferritin H subunit gene is specifically expressed in white pigment cells, but not in melanophores. Pigment organellogenesis and specific gene expression in white pigment cells are also discussed.

  18. Long-term culture and differentiation of CNS precursors derived from anterior human neural rosettes following exposure to ventralizing factors

    SciTech Connect

    Colleoni, Silvia; Giannelli, Serena G.; Armentero, Marie-Therese; Blandini, Fabio; Broccoli, Vania; Lazzari, Giovanna

    2010-04-15

    In this study we demonstrated that neural rosettes derived from human ES cells can give rise either to neural crest precursors, following expansion in presence of bFGF and EGF, or to dopaminergic precursors after exposure to ventralizing factors Shh and FGF8. Both regionalised precursors are capable of extensive proliferation and differentiation towards the corresponding terminally differentiated cell types. In particular, peripheral neurons, cartilage, bone, smooth muscle cells and also pigmented cells were obtained from neural crest precursors while tyrosine hydroxylase and Nurr1 positive dopaminergic neurons were derived from FGF8 and Shh primed rosette cells. Gene expression and immunocytochemistry analyses confirmed the expression of dorsal and neural crest genes such as Sox10, Slug, p75, FoxD3, Pax7 in neural precursors from bFGF-EGF exposed rosettes. By contrast, priming of rosettes with FGF8 and Shh induced the expression of dopaminergic markers Engrailed1, Pax2, Pitx3, floor plate marker FoxA2 and radial glia markers Blbp and Glast, the latter in agreement with the origin of dopaminergic precursors from floor plate radial glia. Moreover, in vivo transplant of proliferating Shh/FGF8 primed precursors in parkinsonian rats demonstrated engraftment and terminal dopaminergic differentiation. In conclusion, we demonstrated the derivation of long-term self-renewing precursors of selected regional identity as potential cell reservoirs for cell therapy applications, such as CNS degenerative diseases, or for the development of toxicological tests.

  19. Whole-cell fungal transformation of precursors into dyes.

    PubMed

    Polak, Jolanta; Jarosz-Wilkołazka, Anna

    2010-07-05

    Chemical methods of producing dyes involve extreme temperatures and unsafe toxic compounds. Application of oxidizing enzymes obtained from fungal species, for example laccase, is an alternative to chemical synthesis of dyes. Laccase can be replaced by fungal biomass acting as a whole-cell biocatalyst with properties comparable to the isolated form of the enzyme. The application of the whole-cell system simplifies the transformation process and reduces the time required for its completion. In the present work, four fungal strains with a well-known ability to produce laccase were tested for oxidation of 17 phenolic and non-phenolic precursors into stable and non-toxic dyes. An agar-plate screening test of the organic precursors was carried out using four fungal strains: Trametes versicolor, Fomes fomentarius, Abortiporus biennis, and Cerrena unicolor. Out of 17 precursors, nine were transformed into coloured substances in the presence of actively growing fungal mycelium. The immobilized fungal biomass catalyzed the transformation of 1 mM benzene and naphthalene derivatives in liquid cultures yielding stable and non-toxic products with good dyeing properties. The type of fungal strain had a large influence on the absorbance of the coloured products obtained after 48-hour transformation of the selected precursors, and the most effective was Fomes fomentarius (FF25). Whole-cell transformation of AHBS (3-amino-4-hydroxybenzenesulfonic acid) into a phenoxazinone dye was carried out in four different systems: in aqueous media comprising low amounts of carbon and nitrogen source, in buffer, and in distilled water. This study demonstrated the ability of four fungal strains belonging to the ecological type of white rot fungi to transform precursors into dyes. This paper highlights the potential of fungal biomass for replacing isolated enzymes as a cheaper industrial-grade biocatalyst for the synthesis of dyes and other commercially important products. The use of immobilized

  20. Use of plankton-derived vitamin B1 precursors, especially thiazole-related precursor, by key marine picoeukaryotic phytoplankton.

    PubMed

    Paerl, Ryan W; Bouget, Francois-Yves; Lozano, Jean-Claude; Vergé, Valérie; Schatt, Philippe; Allen, Eric E; Palenik, Brian; Azam, Farooq

    2017-03-01

    Several cosmopolitan marine picoeukaryotic phytoplankton are B1 auxotrophs requiring exogenous vitamin B1 or precursor to survive. From genomic evidence, representatives of picoeukaryotic phytoplankton (Ostreococcus and Micromonas spp.) were predicted to use known thiazole and pyrimidine B1 precursors to meet their B1 demands, however, recent culture-based experiments could not confirm this assumption. We hypothesized these phytoplankton strains could grow on precursors alone, but required a thiazole-related precursor other the well-known and extensively tested 4-methyl-5-thiazoleethanol. This hypothesis was tested using bioassays and co-cultures of picoeukaryotic phytoplankton and bacteria. We found that specific B1-synthesizing proteobacteria and phytoplankton are sources of a yet-to-be chemically identified thiazole-related precursor(s) that, along with pyrimidine B1 precursor 4-amino-5-hydroxymethyl-2-methylpyrimidine, can support growth of Ostreococcus spp. (also Micromonas spp.) without B1. We additionally found that the B1-synthesizing plankton do not require contact with picoeukaryotic phytoplankton cells to produce thiazole-related precursor(s). Experiments with wild-type and genetically engineered Ostreococcus lines revealed that the thiazole kinase, ThiM, is required for growth on precursors, and that thiazole-related precursor(s) accumulate to appreciable levels in the euphotic ocean. Overall, our results point to thiazole-related B1 precursors as important micronutrients promoting the survival of abundant phytoplankton influencing surface ocean production and biogeochemical cycling.

  1. Use of plankton-derived vitamin B1 precursors, especially thiazole-related precursor, by key marine picoeukaryotic phytoplankton

    PubMed Central

    Paerl, Ryan W; Bouget, Francois-Yves; Lozano, Jean-Claude; Vergé, Valérie; Schatt, Philippe; Allen, Eric E; Palenik, Brian; Azam, Farooq

    2017-01-01

    Several cosmopolitan marine picoeukaryotic phytoplankton are B1 auxotrophs requiring exogenous vitamin B1 or precursor to survive. From genomic evidence, representatives of picoeukaryotic phytoplankton (Ostreococcus and Micromonas spp.) were predicted to use known thiazole and pyrimidine B1 precursors to meet their B1 demands, however, recent culture-based experiments could not confirm this assumption. We hypothesized these phytoplankton strains could grow on precursors alone, but required a thiazole-related precursor other the well-known and extensively tested 4-methyl-5-thiazoleethanol. This hypothesis was tested using bioassays and co-cultures of picoeukaryotic phytoplankton and bacteria. We found that specific B1-synthesizing proteobacteria and phytoplankton are sources of a yet-to-be chemically identified thiazole-related precursor(s) that, along with pyrimidine B1 precursor 4-amino-5-hydroxymethyl-2-methylpyrimidine, can support growth of Ostreococcus spp. (also Micromonas spp.) without B1. We additionally found that the B1-synthesizing plankton do not require contact with picoeukaryotic phytoplankton cells to produce thiazole-related precursor(s). Experiments with wild-type and genetically engineered Ostreococcus lines revealed that the thiazole kinase, ThiM, is required for growth on precursors, and that thiazole-related precursor(s) accumulate to appreciable levels in the euphotic ocean. Overall, our results point to thiazole-related B1 precursors as important micronutrients promoting the survival of abundant phytoplankton influencing surface ocean production and biogeochemical cycling. PMID:27935586

  2. Decreased neural precursor cell pool in NADPH oxidase 2-deficiency: From mouse brain to neural differentiation of patient derived iPSC.

    PubMed

    Nayernia, Zeynab; Colaianna, Marilena; Robledinos-Antón, Natalia; Gutzwiller, Eveline; Sloan-Béna, Frédérique; Stathaki, Elisavet; Hibaoui, Yousef; Cuadrado, Antonio; Hescheler, Jürgen; Stasia, Marie-José; Saric, Tomo; Jaquet, Vincent; Krause, Karl-Heinz

    2017-10-01

    There is emerging evidence for the involvement of reactive oxygen species (ROS) in the regulation of stem cells and cellular differentiation. Absence of the ROS-generating NADPH oxidase NOX2 in chronic granulomatous disease (CGD) patients, predominantly manifests as immune deficiency, but has also been associated with decreased cognition. Here, we investigate the role of NOX enzymes in neuronal homeostasis in adult mouse brain and in neural cells derived from human induced pluripotent stem cells (iPSC). High levels of NOX2 were found in mouse adult neurogenic regions. In NOX2-deficient mice, neurogenic regions showed diminished redox modifications, as well as decrease in neuroprecursor numbers and in expression of genes involved in neural differentiation including NES, BDNF and OTX2. iPSC from healthy subjects and patients with CGD were used to study the role of NOX2 in human in vitro neuronal development. Expression of NOX2 was low in undifferentiated iPSC, upregulated upon neural induction, and disappeared during neuronal differentiation. In human neurospheres, NOX2 protein and ROS generation were polarized within the inner cell layer of rosette structures. NOX2 deficiency in CGD-iPSCs resulted in an abnormal neural induction in vitro, as revealed by a reduced expression of neuroprogenitor markers (NES, BDNF, OTX2, NRSF/REST), and a decreased generation of mature neurons. Vector-mediated NOX2 expression in NOX2-deficient iPSCs rescued neurogenesis. Taken together, our study provides novel evidence for a regulatory role of NOX2 during early stages of neurogenesis in mouse and human. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  3. Follicular Dendritic Cells Emerge from Ubiquitous Perivascular Precursors

    PubMed Central

    Krautler, Nike Julia; Kana, Veronika; Kranich, Jan; Tian, Yinghua; Perera, Dushan; Lemm, Doreen; Schwarz, Petra; Armulik, Annika; Browning, Jeffrey L.; Tallquist, Michelle; Buch, Thorsten; Oliveira-Martins, José B.; Zhu, Caihong; Hermann, Mario; Wagner, Ulrich; Brink, Robert; Heikenwalder, Mathias; Aguzzi, Adriano

    2013-01-01

    Summary The differentiation of follicular dendritic cells (FDC) is essential to the remarkable microanatomic plasticity of lymphoid follicles. Here we show that FDC arise from ubiquitous perivascular precursors (preFDC) expressing platelet-derived growth factor receptor β (PDGFRβ). PDGFRβ-Cre-driven reporter gene recombination resulted in FDC labeling, whereas conditional ablation of PDGFRβ+-derived cells abolished FDC, indicating that FDC originate from PDGFRβ+ cells. Lymphotoxin-α-overexpressing prion protein (PrP)+ kidneys developed PrP+ FDC after transplantation into PrP mice, confirming that preFDC exist outside lymphoid organs. Adipose tissue-derived PDGFRβ+ stromal-vascular cells responded to FDC maturation factors and, when transplanted into lymphotoxin β receptor (LTβR) kidney capsules, differentiated into Mfge8+CD21/35+ FcγRIIβ+PrP+ FDC capable of trapping immune complexes and recruiting B cells. Spleens of lymphocyte-deficient mice contained perivascular PDGFRβ+ FDC precursors whose expansion required both lymphoid tissue inducer (LTi) cells and lymphotoxin. The ubiquity of preFDC and their strategic location at blood vessels may explain the de novo generation of organized lymphoid tissue at sites of lymphocytic inflammation. PMID:22770220

  4. Anti-melanoma vaccinal capacity of CD11c-positive and -negative cell populations present in GM-CSF cultures derived from murine bone marrow precursors.

    PubMed

    Campisano, Sabrina; Mac Keon, Soledad; Gazzaniga, Silvina; Ruiz, María Sol; Traian, Martín Dodes; Mordoh, José; Wainstok, Rosa

    2013-01-02

    We have initially shown that DC/ApoNec vaccine can induce protection against the poorly immunogenic B16F1 melanoma in mice. The population of DC obtained for vaccination after 7days culture with murine GM-CSF is heterogeneous and presents about 60% of CD11c+ DC. Therefore, our purpose was to identify the phenotype of the cells obtained after differentiation and its immunogenicity once injected. DC were separated with anti-CD11c microbeads and the two populations identified in terms of CD11c positivity (DC+ and DC-) were also studied. Approximately 26.6% of the cells in DC+ fraction co-expressed CD11c+ and F4/80 markers and 75.4% were double positive for CD11c and CD11b markers. DC+ fraction also expressed Ly6G. DC- fraction was richer in CD11c-/F4/80+ macrophages (44.7%), some of which co-expressed Ly6G (41.8%), and F4/80-/Ly6-G+ neutrophils (34.6%). Both DC+ and DC- fractions displayed similar capacity to phagocyte and endocyte antigens and even expressed levels of MHC Class II and CD80, CD83 and CD86 costimulatory molecules similar to those in the DC fraction. However, only DC/ApoNec vaccine was capable to induce protection in mice (p<0.01). After 24h co-culture, no detectable level of IL-12 was recorded in DC/ApoNec vaccine, either in supernatant or intracellularly. Therefore, the protection obtained with DC/ApoNec vaccine seemed to be independent of the vaccine's ability to secrete this inflammatory cytokine at the time of injection. In conclusion, we demonstrated that all cell types derived from the culture of mouse bone marrow with GM-CSF are necessary to induce antitumor protection in vivo. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Differentiation of human adult skin-derived neuronal precursors into mature neurons.

    PubMed

    Gingras, Marie; Champigny, Marie-France; Berthod, François

    2007-02-01

    The isolation of autologous neuronal precursors from skin-derived precursor cells extracted from adult human skin would be a very efficient source of neurons for the treatment of various neurodegenerative diseases. The purpose of this study was to demonstrate that these neuronal precursors were able to differentiate into mature neurons. We isolated neuronal precursors from breast skin and expanded them in vitro for over ten passages. We showed that 48% of these cells were proliferating after the first passage, while this growth rate decreased after the second passage. We demonstrated that 70% of these cells were nestin-positive after the third passage, while only 17% were neurofilament M-positive after 7 days of differentiation. These neuronal precursors expressed betaIII tubulin, the dendritic marker MAP2 and the presynaptic marker synaptophysin after 7 days of in vitro maturation. They also expressed the postsynaptic marker PSD95 and the late neuronal markers NeuN and neurofilament H after 21 days of differentiation, demonstrating they became terminally differentiated neurons. These markers were still expressed after 50 days of culture. The generation of autologous neurons from an accessible adult human source opens many potential therapeutic applications and has a great potential for the development of experimental studies on normal human neurons.

  6. Electrochemical behavior of carbon aerogels derived from different precursors

    SciTech Connect

    Pekala, R.W.; Alviso, C.T.; Nielson, J.K.; Tran, T.D.; Reynolds, G.M.; Dresshaus, M.S.

    1995-04-01

    The ability to tailor the structure and properties of porous carbons has led to their increased use as electrodes in energy storage devices. Our research focuses on the synthesis and characterization of carbon aerogels for use in electrochemical double layer capacitors. Carbon aerogels are formed from the sol-gel polymerization of (1) resorcinol-formaldehyde or (2) phenolic-furfural, followed by supercritical drying from carbon dioxide, and subsequent pyrolysis in an inert atmosphere. These materials can be produced as monoliths, composites, thin films, powders, or microspheres. In all cases, the areogels have an open-cell structure with an ultrafine pore size (<100 nm), high surface area (400-1 100 m{sup 2}/g), and a solid matrix composed of interconnected particles, fibers, or platelets with characteristic dimensions of 10 nm. This paper examines the effects of the carbon precursor and processing conditions on electrochemical performance in aqueous and organic electrolytes.

  7. Electrochemical behavior of carbon aerogels derived from different precursors

    SciTech Connect

    Pekala, R.W.; Alviso, C.T.; Nielsen, J.K.; Tran, T.D.; Reynolds, G.A.M.; Dresselhaus, M.S.

    1995-12-31

    The ability to tailor the structure and properties of porous carbons has led to their increased use as electrodes in energy storage devices. The research focuses on the synthesis and characterization of carbon aerogels for use in electrochemical double layer capacitors.Carbon aerogels are formed from the sol-gel polymerization of (1) resorcinol-formaldehyde or (2) phenolic-furfural, followed by supercritical drying from carbon dioxide, and subsequent pyrolysis in an inert atmosphere. These materials can be produced as monoliths, composites, thin films, powders, or microspheres. In all cases, the aerogels have an open-cell structure with an ultrafine pore size (< 100 nm), high surface area (400--1,100 m{sup 2}/g), and a solid matrix composed of interconnected particles, fibers, or platelets with characteristic dimensions of 10 nm. This paper examines the effects of the carbon precursor and processing conditions on electrochemical performance in aqueous and organic electrolytes.

  8. Long-term fate of neural precursor cells following transplantation into developing and adult CNS.

    PubMed

    Lepore, A C; Neuhuber, B; Connors, T M; Han, S S W; Liu, Y; Daniels, M P; Rao, M S; Fischer, I

    2006-05-12

    Successful strategies for transplantation of neural precursor cells for replacement of lost or dysfunctional CNS cells require long-term survival of grafted cells and integration with the host system, potentially for the life of the recipient. It is also important to demonstrate that transplants do not result in adverse outcomes. Few studies have examined the long-term properties of transplanted neural precursor cells in the CNS, particularly in non-neurogenic regions of the adult. The aim of the present study was to extensively characterize the fate of defined populations of neural precursor cells following transplantation into the developing and adult CNS (brain and spinal cord) for up to 15 months, including integration of graft-derived neurons with the host. Specifically, we employed neuronal-restricted precursors and glial-restricted precursors, which represent neural precursor cells with lineage restrictions for neuronal and glial fate, respectively. Transplanted cells were prepared from embryonic day-13.5 fetal spinal cord of transgenic donor rats that express the marker gene human placental alkaline phosphatase to achieve stable and reliable graft tracking. We found that in both developing and adult CNS grafted cells showed long-term survival, morphological maturation, extensive distribution and differentiation into all mature CNS cell types (neurons, astrocytes and oligodendrocytes). Graft-derived neurons also formed synapses, as identified by electron microscopy, suggesting that transplanted neural precursor cells integrated with adult CNS. Furthermore, grafts did not result in any apparent deleterious outcomes. We did not detect tumor formation, cells did not localize to unwanted locations and no pronounced immune response was present at the graft sites. The long-term stability of neuronal-restricted precursors and glial-restricted precursors and the lack of adverse effects suggest that transplantation of lineage-restricted neural precursor cells can

  9. Whole-cell fungal transformation of precursors into dyes

    PubMed Central

    2010-01-01

    Background Chemical methods of producing dyes involve extreme temperatures and unsafe toxic compounds. Application of oxidizing enzymes obtained from fungal species, for example laccase, is an alternative to chemical synthesis of dyes. Laccase can be replaced by fungal biomass acting as a whole-cell biocatalyst with properties comparable to the isolated form of the enzyme. The application of the whole-cell system simplifies the transformation process and reduces the time required for its completion. In the present work, four fungal strains with a well-known ability to produce laccase were tested for oxidation of 17 phenolic and non-phenolic precursors into stable and non-toxic dyes. Results An agar-plate screening test of the organic precursors was carried out using four fungal strains: Trametes versicolor, Fomes fomentarius, Abortiporus biennis, and Cerrena unicolor. Out of 17 precursors, nine were transformed into coloured substances in the presence of actively growing fungal mycelium. The immobilized fungal biomass catalyzed the transformation of 1 mM benzene and naphthalene derivatives in liquid cultures yielding stable and non-toxic products with good dyeing properties. The type of fungal strain had a large influence on the absorbance of the coloured products obtained after 48-hour transformation of the selected precursors, and the most effective was Fomes fomentarius (FF25). Whole-cell transformation of AHBS (3-amino-4-hydroxybenzenesulfonic acid) into a phenoxazinone dye was carried out in four different systems: in aqueous media comprising low amounts of carbon and nitrogen source, in buffer, and in distilled water. Conclusions This study demonstrated the ability of four fungal strains belonging to the ecological type of white rot fungi to transform precursors into dyes. This paper highlights the potential of fungal biomass for replacing isolated enzymes as a cheaper industrial-grade biocatalyst for the synthesis of dyes and other commercially important

  10. Innate lymphoid cells, precursors and plasticity.

    PubMed

    Gronke, Konrad; Kofoed-Nielsen, Michael; Diefenbach, Andreas

    2016-11-01

    Innate lymphoid cells (ILC) have only recently been recognized as a separate entity of the lymphoid lineage. Their subpopulations share common characteristics in terms of early development and major transcriptional circuitry with their related cousins of the T cell world. It is currently hypothesized that ILCs constitute an evolutionary older version of the lymphoid immune system. They are found at all primary entry points for pathogens such as mucosal surfaces of the lung and gastrointestinal system, the skin and the liver, which is the central contact point for pathogens that breach the intestinal barrier and enter the circulation. There, ILC contribute to the first line defense as well as to organ homeostasis. However, ILC are not only involved in classical defense tasks, but also contribute to the organogenesis of lymphoid organs as well as tissue remodeling and even stem cell regeneration. ILC may, therefore, implement different functions according to their emergence in ontogeny, their development and their final tissue location. We will review here their early development from precursors of the fetal liver and the adult bone marrow as well as their late plasticity in adaptation to their environment. Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  11. Auraptene induces oligodendrocyte lineage precursor cells in a cuprizone-induced animal model of demyelination.

    PubMed

    Nakajima, Mitsunari; Shimizu, Risei; Furuta, Kohei; Sugino, Mami; Watanabe, Takashi; Aoki, Rui; Okuyama, Satoshi; Furukawa, Yoshiko

    2016-05-15

    We investigated the effects of auraptene on mouse oligodendroglial cell lineage in an animal model of demyelination induced by cuprizone. Auraptene, a citrus coumarin, was intraperitoneally administered to mice fed the demyelinating agent cuprizone. Immunohistochemical analysis of the corpus callosum and/or Western blotting analysis of brain extracts revealed that cuprizone reduced immunoreactivity for myelin-basic protein, a marker of myelin, whereas it increased immunoreactivity to platelet derived-growth factor receptor-α, a marker of oligodendrocyte precursor cells. Administration of auraptene enhanced the immunoreactivity to oligodendrocyte transcription factor 2, a marker of oligodendrocyte precursor cells and oligodendrocyte lineage precursor cells, but had no effect on immunoreactivity to myelin-basic protein or platelet-derived growth factor receptor-α. These findings suggest that auraptene promotes the production of oligodendrocyte lineage precursor cells in an animal model of demyelination and may be useful for individuals with demyelinating diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Glial versus melanocyte cell fate choice: Schwann cell precursors as a cellular origin of melanocytes.

    PubMed

    Adameyko, Igor; Lallemend, Francois

    2010-09-01

    Melanocytes and Schwann cells are derived from the multipotent population of neural crest cells. Although both cell types were thought to be generated through completely distinct pathways and molecular processes, a recent study has revealed that these different cell types are intimately interconnected far beyond previously postulated limits in that they share a common post-neural crest progenitor, i.e. the Schwann cell precursor. This finding raises interesting questions about the lineage relationships of hitherto unrelated cell types such as melanocytes and Schwann cells, and may provide clinical insights into mechanisms of pigmentation disorders and for cancer involving Schwann cells and melanocytes.

  13. Sustainable Life Cycles of Natural-Precursor-Derived Nanocarbons.

    PubMed

    Bazaka, Kateryna; Jacob, Mohan V; Ostrikov, Kostya Ken

    2016-01-13

    Sustainable societal and economic development relies on novel nanotechnologies that offer maximum efficiency at minimal environmental cost. Yet, it is very challenging to apply green chemistry approaches across the entire life cycle of nanotech products, from design and nanomaterial synthesis to utilization and disposal. Recently, novel, efficient methods based on nonequilibrium reactive plasma chemistries that minimize the process steps and dramatically reduce the use of expensive and hazardous reagents have been applied to low-cost natural and waste sources to produce value-added nanomaterials with a wide range of applications. This review discusses the distinctive effects of nonequilibrium reactive chemistries and how these effects can aid and advance the integration of sustainable chemistry into each stage of nanotech product life. Examples of the use of enabling plasma-based technologies in sustainable production and degradation of nanotech products are discussed-from selection of precursors derived from natural resources and their conversion into functional building units, to methods for green synthesis of useful naturally degradable carbon-based nanomaterials, to device operation and eventual disintegration into naturally degradable yet potentially reusable byproducts.

  14. BACE1- and BACE2-expressing human cells: characterization of beta-amyloid precursor protein-derived catabolites, design of a novel fluorimetric assay, and identification of new in vitro inhibitors.

    PubMed

    Andrau, David; Dumanchin-Njock, Cecile; Ayral, Erwan; Vizzavona, Jean; Farzan, Mike; Boisbrun, Michel; Fulcrand, Pierre; Hernandez, Jean-Francois; Martinez, Jean; Lefranc-Jullien, Solveig; Checler, Frederic

    2003-07-11

    We have set up stably transfected HEK293 cells overexpressing the beta-secretases BACE1 and BACE2 either alone or in combination with wild-type beta-amyloid precursor protein (betaAPP). The characterization of the betaAPP-derived catabolites indicates that cells expressing BACEs produce less genuine Abeta1- 40/42 but higher amounts of secreted sAPPbeta and N-terminal-truncated Abeta species. This was accompanied by a concomitant modulation of the C-terminal counterpart products C89 and C79 for BACE1 and BACE2, respectively. These cells were used to set up a novel BACE assay based on two quenched fluorimetric substrates mimicking the wild-type (JMV2235) and Swedish-mutated (JMV2236) betaAPP sequences targeted by BACE activities. We show that BACEs activities are enhanced by the Swedish mutation and maximal at pH 4.5. The specificity of this double assay for genuine beta-secretase activity was demonstrated by means of cathepsin D, a "false positive" BACE candidate. Thus, cathepsin D was unable to cleave preferentially the JMV2236-mutated substrate. The selectivity of the assay was also emphasized by the lack of JMV cleavage triggered by other "secretases" candidates such as ADAM10 (A disintegrin and metalloprotease 10), tumor necrosis alpha-converting enzyme, and presenilins 1 and 2. Finally, the assay was used to screen for putative in vitro BACE inhibitors. We identified a series of statine-derived sequences that dose-dependently inhibited BACE1 and BACE2 activities with IC50 in the micromolar range, some of which displaying selectivity for either BACE1 or BACE2.

  15. Different radiosensitivities of mast-cell precursors in the bone marrow and skin of mice

    SciTech Connect

    Kitamura, Y.; Yokoyama, M.; Sonoda, T.; Mori, K.J.

    1983-01-01

    Although tissue mast cells are derived from the bone marrow, some descendants of bone marrow-derived precursors retain the ability to proliferate and differentiate into mast cells even after localization in the skin. The purpose of the present study was to determine the D/sub 0/ values for mast-cell precursors in the bone marrow and those localized in the skin. Bone marrow cells were removed from (WB X C57BL/6)F/sub 1/+/+ mice after various doses of irradiation and injected into the skin of the congenic W/W/sup v/ mice which were genetically without mast cells. Radiosensitivity of mast-cell precursors in the bone marrow was evaluated by determining the proportion of the injection sites at which mast cells did not appear. For the assay of the radiosensitivity of mast-cell precursors localized in the skin, pieces of skin were removed from beige C57BL/6 (bg/sup J//bg/sup J/, Chediak-Higashi syndrome) mice after various doses of irradiation and grafted onto the backs of the normal C57BL/6 mice. Radiosensitivity of mast-cell precursors in the skin was evaluated by determining the decrease of beige-type mast cells which possessed giant granules. Mast-cell precursors in the bone marrow were much more radiosenitive than those localized in the skin. D/sup 0/ value was about 100 rad for the former and about 800 rad for the latter.

  16. Different radiosensitivities of mast-cell precursors in the bone marrow and skin of mice

    SciTech Connect

    Kitamura, Y.; Yokoyama, M.; Sonoda, T.; Mori, K.J.

    1983-01-01

    Although tissue mast cells are derived from the bone marrow, some descendants of bone marrow-derived precursors retain the ability to proliferate and differentiate into mast cells even after localization in the skin. The purpose of the present study was to determine the D0 values for mast-cell precursors in the bone marrow and those localized in the skin. Bone marrow cells were removed from (WB X C57BL/6)F1-+/+ mice after various doses of irradiation and injected into the skin of the congenic W/Wv mice which were genetically without mast cells. Radiosensitivity of mast-cell precursors in the bone marrow was evaluated by determining the proportion of the injection sites at which mast cells did not appear. For the assay of the radiosensitivity of mast-cell precursors localized in the skin, pieces of skin were removed from beige C57BL/6 (bgJ/bgJ. Chediak-Higashi syndrome) mice after various doses of irradiation and grafted onto the back of the normal C57BL/6 mice. Radiosensitivity of mast-cell precursors in the skin was evaluated by determining the decrease of beige-type mast cells which possessed giant granules. Mast-cell precursors in the bone marrow were much more radiosensitive than those localized in the skin. D0 value was about 100 rad for the former and about 800 rad for the latter.

  17. Epithelial expression of extracellular matrix metalloproteinase inducer/CD147 and matrix metalloproteinase-2 in neoplasms and precursor lesions derived from cutaneous squamous cells: An immunohistochemical study.

    PubMed

    Ayva, Sebnem Kupana; Karabulut, Ayse Anil; Akatli, Ayşe Nur; Atasoy, Pinar; Bozdogan, Onder

    2013-10-01

    Extracellular matrix metalloproteinase inducer (CD147) is a transmembrane glycoprotein involved in the regulation of matrix metalloproteinases (MMPs). The study investigated CD147 and MMP-2 expression in epidermis of cutaneous squamous lesions. CD147 and MMP-2 expressions were evaluated immunohistochemically in 44 specimens: 18 actinic keratoses (AK), 6 squamous cell carcinomas in situ (SCCIS), 13 squamous cell carcinomas (SCC; peritumoral and invasive portions assessed), and 7 normal skins. Patterns of expression were assessed, with MMP-2 in nuclei (MMP-2n) and cytoplasm (MMP-2c) evaluated separately. The expression of each marker was quantified using a calculated immunohistochemical/histologic score (H-score). Correlations were analyzed for the marker H-scores in each study group. Associations between H-scores and histopathologic parameters were also evaluated. CD147 H-score was the highest in SCC (invasive islands), followed by AK, SCCIS, and control specimens, respectively. MMP-2n and MMP-2c H-scores were the highest in AK, followed by SCCIS, SCC, and control specimens, respectively. MMP-2c and MMP-2n H-scores were significantly higher in peritumoral epidermis than in invasive islands of SCC. MMP-2c and CD147 H-scores were positively correlated in the peritumoral SCCs. CD147 H-score was positively correlated with tumor differentiation in SCC. The findings suggest that overexpression of CD147 plays a role in the development of SCC.

  18. Morphine Withdrawal Increases Brain-Derived Neurotrophic Factor Precursor.

    PubMed

    Bachis, Alessia; Campbell, Lee A; Jenkins, Kierra; Wenzel, Erin; Mocchetti, Italo

    2017-08-03

    Morphine has been shown to increase the expression of brain-derived neurotrophic factor (BDNF) in the brain. However, little is known about the effect of morphine withdrawal on BDNF and its precursor protein, or proBDNF, which induces neuronal apoptosis. In this work, we examined whether BDNF and proBDNF levels change in rats chronically injected with escalating doses of morphine and those who undergo spontaneous withdrawal for 60 h. We observed, in the frontal cortex and striatum, that the ratio of BDNF to proBDNF changed depending upon the experimental paradigm. Morphine treatment and morphine withdrawal increased both BDNF and proBDNF levels. However, the increase in proBDNF immunoreactivity in withdrawal rats was more robust than that observed in morphine-treated rats. proBDNF is processed either intracellularly by furin or extracellularly by the tissue plasminogen activator (tPA)/plasminogen system or matrix metalloproteases (MMPs). To examine the mechanisms whereby chronic morphine treatment and morphine withdrawal differentially affects BDNF/proBDNF, the levels MMP-3 and MMP-7, furin, and tPA were analyzed. We found that morphine increases tPA levels, whereas withdrawal causes a decrease. To confirm the involvement of tPA in the morphine-mediated effect on BDNF/proBDNF, we exposed cortical neurons to morphine in the presence of the tPA inhibitor plasminogen activator inhibitor-1 (PAI-1). This inhibitor reversed the morphine-mediated decrease in proBDNF, supporting the hypothesis that morphine increases the availability of BDNF by promoting the extracellular processing of proBDNF by tPA. Because proBDNF could negatively influence synaptic repair, preventing withdrawal is crucial for reducing neurotoxic mechanisms associated with opioid abuse.

  19. Mechanisms of gravitational sensitivity of osteogenic precursor cells.

    PubMed

    Buravkova, L B; Gershovich, P M; Gershovich, J G; Grigor'ev, A I

    2010-04-01

    This report is a detailed review of the current data on the mechanic and gravitational sensitivity of osteoblasts and osteogenic precursor cells in vitro. It summarizes the numerous responses of cells with an osteoblastic phenotype and osteogenic precursor cells and especially their responses to the alteration of their mechanic or gravitational surroundings. The review also discusses the osteogenic cell's pathways of signal transduction and the mechanisms of gravitational sensitivity. It was shown that the earliest multipotent stromal precursor cells of an adult organism's bone marrow can sense changes of intensity in a gravitational or mechanic field in model conditions, which may play a certain role in the development of osteopenia in microgravity.

  20. A primitive cell origin for B-cell precursor ALL?

    PubMed

    Cox, C V; Blair, A

    2005-01-01

    A stem cell origin has been described for both acute and chronic myelogenous leukemias. In contrast, childhood B-cell precursor acute lymphoblastic leukemia (ALL) is thought to arise in committed B-lineage cells. Recently described in vitro and in vivo model systems that support the proliferation and expansion of ALL cells have provided new tools to investigate the cellular targets for the origin of this malignancy. Evidence suggests that some subtypes of childhood ALL have a primitive cell origin and share many immunophenotypic characteristics with normal progenitor cells. These leukemic stem cells may be resistant to current therapeutic strategies designed to kill the bulk ALL cell population and subsequent relapses may arise from this population. More precise definition of these ALL stem cells through combined analyses of antigen expression, genetic lesions, and functionality is essential for the development of more effective, targeted therapeutic strategies.

  1. Doublecortin in Oligodendrocyte Precursor Cells in the Adult Mouse Brain

    PubMed Central

    Boulanger, Jenna J.; Messier, Claude

    2017-01-01

    Key Points Oligodendrocyte precursor cells express doublecortin, a microtubule-associated protein.Oligodendrocyte precursor cells express doublecortin, but at a lower level of expression than in neuronal precursor.Doublecortin is not associated with a potential immature neuronal phenotype in Oligodendrocyte precursor cells. Oligodendrocyte precursor cells (OPC) are glial cells that differentiate into myelinating oligodendrocytes during embryogenesis and early stages of post-natal life. OPCs continue to divide throughout adulthood and some eventually differentiate into oligodendrocytes in response to demyelinating lesions. There is growing evidence that OPCs are also involved in activity-driven de novo myelination of previously unmyelinated axons and myelin remodeling in adulthood. Considering these roles in the adult brain, OPCs are likely mobile cells that can migrate on some distances before they differentiate into myelinating oligodendrocytes. A number of studies have noted that OPCs express doublecortin (DCX), a microtubule-associated protein expressed in neural precursor cells and in migrating immature neurons. Here we describe the distribution of DCX in OPCs. We found that almost all OPCs express DCX, but the level of expression appears to be much lower than what is found in neural precursor. We found that DCX is downregulated when OPCs start expressing mature oligodendrocyte markers and is absent in myelinating oligodendrocytes. DCX does not appear to signal an immature neuronal phenotype in OPCs in the adult mouse brain. Rather, it could be involved either in cell migration, or as a marker of an immature oligodendroglial cell phenotype. PMID:28400715

  2. Diversity of Neural Precursor Cell Types in the Prenatal Macaque Cerebral Cortex Exists Largely within the Astroglial Cell Lineage

    PubMed Central

    Cunningham, Christopher L.; Martínez-Cerdeño, Verónica; Noctor, Stephen C.

    2013-01-01

    The germinal zones of the embryonic macaque neocortex comprise the ventricular zone (VZ) and the subventricular zone (SVZ). The mammalian SVZ is subdivided into an inner SVZ and an outer SVZ, with the outer SVZ being particularly large in primates. The existence of distinct precursor cell types in the neocortical proliferative zones was inferred over 100 years ago and recent evidence supports this concept. Precursor cells exhibiting diverse morphologies, patterns of transcription factor expression, and fate potential have been identified in the neocortical proliferative zones. Neurogenic precursor cells are thought to exhibit characteristics of glial cells, but the existence of neurogenic precursor cells that do not share glial specific properties has also been proposed. Therefore, one question that remains is whether neural precursor cells in the prenatal neocortex belong within the astroglial cell class, as they do in neurogenic regions of the adult neocortex, or instead include a diverse collection of precursor cells belonging to distinct cell classes. We examined the expression of astroglial markers by mitotic precursor cells in the telencephalon of prenatal macaque and human. We show that in the dorsal neocortex all mitotic cells at the surface of the ventricle, and all Pax6+ and Tbr2+ mitotic cells in the proliferative zones, express the astroglial marker GFAP. The majority of mitotic cells undergoing division away from the ventricle express GFAP, and many of the GFAP-negative mitoses express markers of cells derived from the ventral telencephalon or extracortical sites. In contrast, a markedly lower proportion of precursor cells express GFAP in the ganglionic eminence. In conclusion, we propose that the heterogeneity of neural precursor cells in the dorsal cerebral cortex develops within the GFAP+ astroglial cell class. PMID:23724007

  3. High Temperature Stability of Binary Microstructures Derived from Liquid Precursors

    DTIC Science & Technology

    1994-06-30

    substrates have bee.t used to produce single crystal ferroelectric films. Sapphire substrates were used for Nb - doped PZT thin films.25 The growth of...Epitaxial Growth of PbTiO3 Thin Films on (100) SrTiO3 from Solution Precursors Andreas Seifert, Fred F. Lange and James S. Speck Technical Report No...12 Abstract: A mixed alkoxide liquid precursor was used to form epitaxial PbTiO3 thin films by spin-coating on cubic (001) SrTiO3 substrates. The films

  4. Lack of evidence for recipient precursor cells replenishing β-cells in transplanted islets.

    PubMed

    Hamamoto, Yoshiyuki; Akashi, Tomoyuki; Inada, Akari; Bonner-Weir, Susan; Weir, Gordon C

    2010-01-01

    Bone marrow and tissue precursor cells have been postulated to replenish grafts of transplanted islets. Several investigators have reported that bone marrow cells can promote the regeneration of injured islets. In this study, we investigated the potential of recipient-derived precursor cells to form new pancreatic endocrine cells in islet grafts transplanted under the kidney capsule. Mouse insulin promoter (MIP)-green fluorescence protein (GFP) mice, which express GFP only in β-cells, or β-actin GFP mice, which express GFP ubiquitously, were used to determine if the recipient-derived cells differentiate into β-cells or other types of endocrine cells. We transplanted MIP-GFP islets into wild-type mice, wild-type islets into MIP-GFP mice, β-actin GFP islets into wild-type mice, and wild-type islets into β-actin GFP mice. β-Actin GFP bone marrow cells were then injected into wild-type mice to evaluate the potential role of bone marrow stem cells to provide new islet cells to the graft. No β-cells with green fluorescence were seen in the graft when wild-type islets were transplanted into MIP-GFP mice. When wild-type islets were transplanted into β-actin GFP mice, no β-cells with GFP staining could be identified in the grafts. Similarly, no endocrine cells with GFP staining could be identified in the grafts after injection of β-actin GFP bone marrow cells into wild-type islet-transplanted wild-type mice. This study provides further support for the concept that recipient precursor cells do not produce new β-cells in grafts of transplanted islets.

  5. Role of neural precursor cells in promoting repair following stroke

    PubMed Central

    Dibajnia, Pooya; Morshead, Cindi M

    2013-01-01

    Stem cell-based therapies for the treatment of stroke have received considerable attention. Two broad approaches to stem cell-based therapies have been taken: the transplantation of exogenous stem cells, and the activation of endogenous neural stem and progenitor cells (together termed neural precursors). Studies examining the transplantation of exogenous cells have demonstrated that neural stem and progenitor cells lead to the most clinically promising results. Endogenous activation of neural precursors has also been explored based on the fact that resident precursor cells have the inherent capacity to proliferate, migrate and differentiate into mature neurons in the uninjured adult brain. Studies have revealed that these neural precursor cell behaviours can be activated following stroke, whereby neural precursors will expand in number, migrate to the infarct site and differentiate into neurons. However, this innate response is insufficient to lead to functional recovery, making it necessary to enhance the activation of endogenous precursors to promote tissue repair and functional recovery. Herein we will discuss the current state of the stem cell-based approaches with a focus on endogenous repair to treat the stroke injured brain. PMID:23064725

  6. Myeloid derived suppressor cells in transplantation.

    PubMed

    Lees, Jason R; Azimzadeh, Agnes M; Bromberg, Jonathan S

    2011-10-01

    Myeloid derived suppressor cells (MDSC) are a heterogeneous population of hematopoietic derived cell precursors that can suppress immune responses in a variety of inflammatory settings. Here we review recent studies detailing expansion of phenotypically and functionally disparate MDSC. Findings related to MDSC accumulation, activation, and mechanisms utilized in immune suppression are presented. Further, we discuss recent reports that suggest MDSC are expanded during transplantation and that modulation of MDSC can participate in preventing graft rejection.

  7. Common precursors for neural and mesectodermal derivatives in the cephalic neural crest.

    PubMed

    Baroffio, A; Dupin, E; Le Douarin, N M

    1991-05-01

    The cephalic neural crest (NC) of vertebrate embryos yields a variety of cell types belonging to the neuronal, glial, melanocytic and mesectodermal lineages. Using clonal cultures of quail migrating cephalic NC cells, we demonstrated that neurons and glial cells of the peripheral nervous system can originate from the same progenitors as cartilage, one of the mesectodermal derivatives of the NC. Moreover, we obtained evidence that the migrating cephalic NC contains a few highly multipotent precursors that are common to neurons, glia, cartilage and pigment cells and which we interprete as representative of a stem cell population. In contrast, other NC cells, although provided with identical culture conditions, give rise to clones composed of only one or some of these cell types. These cells thus appear restricted in their developmental potentialities compared to multipotent cells. It is therefore proposed that, in vivo, the active proliferation of pluripotent NC cells during the migration process generates distinct subpopulations of cells that become progressively committed to different developmental fates.

  8. Dopamine depletion impairs precursor cell proliferation in Parkinson disease.

    PubMed

    Höglinger, Günter U; Rizk, Pamela; Muriel, Marie P; Duyckaerts, Charles; Oertel, Wolfgang H; Caille, Isabelle; Hirsch, Etienne C

    2004-07-01

    Cerebral dopamine depletion is the hallmark of Parkinson disease. Because dopamine modulates ontogenetic neurogenesis, depletion of dopamine might affect neural precursors in the subependymal zone and subgranular zone of the adult brain. Here we provide ultrastructural evidence showing that highly proliferative precursors in the adult subependymal zone express dopamine receptors and receive dopaminergic afferents. Experimental depletion of dopamine in rodents decreases precursor cell proliferation in both the subependymal zone and the subgranular zone. Proliferation is restored completely by a selective agonist of D2-like (D2L) receptors. Experiments with neural precursors from the adult subependymal zone grown as neurosphere cultures confirm that activation of D2L receptors directly increases the proliferation of these precursors. Consistently, the numbers of proliferating cells in the subependymal zone and neural precursor cells in the subgranular zone and olfactory bulb are reduced in postmortem brains of individuals with Parkinson disease. These observations suggest that the generation of neural precursor cells is impaired in Parkinson disease as a consequence of dopaminergic denervation.

  9. Aniline is an inducer, and not a precursor, for indole derivatives in Rubrivivax benzoatilyticus JA2.

    PubMed

    Mujahid, Mohammed; Sasikala, Ch; Ramana, Ch V

    2014-01-01

    Rubrivivax benzoatilyticus JA2 and other anoxygenic photosynthetic bacteria produce indole derivatives when exposed to aniline, a xenobiotic compound. Though this phenomenon has been reported previously, the role of aniline in the production of indoles is still a biochemical riddle. The present study aims at understanding the specific role of aniline (as precursor or stimulator) in the production of indoles and elucidating the biochemical pathway of indoles in aniline-exposed cells by using stable isotope approaches. Metabolic profiling revealed tryptophan accumulation only in aniline exposed cells along with indole 3-acetic acid (IAA) and indole 3-aldehyde (IAld), the two major catabolites of tryptophan. Deuterium labelled aniline feeding studies revealed that aniline is not a precursor of indoles in strain JA2. Further, production of indoles only in aniline-exposed cells suggests that aniline is an indoles stimulator. In addition, production of indoles depended on the presence of a carbon source, and production enhanced when carbon sources were added to the culture. Isotope labelled fumarate feeding identified, fumarate as the precursor of indole, indicating de novo synthesis of indoles. Glyphosate (shikimate pathway inhibitor) inhibited the indoles production, accumulation of tryptophan, IAA and IAld indicating that indoles synthesis in strain JA2 occurs via the de novo shikimate pathway. The up-regulation of anthranilate synthase gene and induction of anthranilate synthase activity correlated well with tryptophan production in strain JA2. Induction of tryptophan aminotransferase and tryptophan 2-monooxygenase activities corroborated well with IAA levels, suggesting that tryptophan catabolism occurs simultaneously in aniline exposed cells. Our study demonstrates that aniline (stress) stimulates tryptophan/indoles synthesis via the shikimate pathway by possibly modulating the metabolic pathway.

  10. Aniline Is an Inducer, and Not a Precursor, for Indole Derivatives in Rubrivivax benzoatilyticus JA2

    PubMed Central

    Mohammed, Mujahid; Ch, Sasikala; Ch, Ramana V.

    2014-01-01

    Rubrivivax benzoatilyticus JA2 and other anoxygenic photosynthetic bacteria produce indole derivatives when exposed to aniline, a xenobiotic compound. Though this phenomenon has been reported previously, the role of aniline in the production of indoles is still a biochemical riddle. The present study aims at understanding the specific role of aniline (as precursor or stimulator) in the production of indoles and elucidating the biochemical pathway of indoles in aniline-exposed cells by using stable isotope approaches. Metabolic profiling revealed tryptophan accumulation only in aniline exposed cells along with indole 3-acetic acid (IAA) and indole 3-aldehyde (IAld), the two major catabolites of tryptophan. Deuterium labelled aniline feeding studies revealed that aniline is not a precursor of indoles in strain JA2. Further, production of indoles only in aniline-exposed cells suggests that aniline is an indoles stimulator. In addition, production of indoles depended on the presence of a carbon source, and production enhanced when carbon sources were added to the culture. Isotope labelled fumarate feeding identified, fumarate as the precursor of indole, indicating de novo synthesis of indoles. Glyphosate (shikimate pathway inhibitor) inhibited the indoles production, accumulation of tryptophan, IAA and IAld indicating that indoles synthesis in strain JA2 occurs via the de novo shikimate pathway. The up-regulation of anthranilate synthase gene and induction of anthranilate synthase activity correlated well with tryptophan production in strain JA2. Induction of tryptophan aminotransferase and tryptophan 2-monooxygenase activities corroborated well with IAA levels, suggesting that tryptophan catabolism occurs simultaneously in aniline exposed cells. Our study demonstrates that aniline (stress) stimulates tryptophan/indoles synthesis via the shikimate pathway by possibly modulating the metabolic pathway. PMID:24533057

  11. Effects of Retinoic Acid Signaling on Extraocular Muscle Myogenic Precursor Cells In Vitro.

    PubMed

    Hebert, Sadie L; Fitzpatrick, Krysta R; McConnell, Samantha A; Cucak, Anja; Yuan, Ching; McLoon, Linda K

    2017-10-07

    One major difference between limb and extraocular muscles (EOM) is the presence of an enriched population of Pitx2-positive myogenic precursor cells in EOM compared to limb muscle. We hypothesize that retinoic acid regulates Pitx2 expression in EOM myogenic precursor cells and that its effects would differ in leg muscle. The two muscle groups expressed differential retinoic acid receptor (RAR) and retinoid X receptor (RXR) levels. RXR co-localized with the Pitx2-positive cells but not with those expressing Pax7. EOM-derived and LEG-derived EECD34 cells were treated with vehicle, retinoic acid, the RAR inverse agonist BMS493, or the RXR antagonist UVI 3003. In vitro, fewer EOM-derived EECD34 cells expressed desmin and fused, while more LEG-derived cells expressed desmin and fused when treated with retinoic acid compared to vehicle. Both EOM and LEG-derived EECD34 cells exposed to retinoic acid showed a higher percentage of cells expressing Pitx2 compared to vehicle, supporting the hypothesis that retinoic acid plays a role in maintaining Pitx2 expression. We hypothesize that retinoic acid signaling aids in the maintenance of large numbers of undifferentiated myogenic precursor cells in the EOM, which would be required to maintain EOM normalcy throughout a lifetime of myonuclear turnover. Copyright © 2017. Published by Elsevier Inc.

  12. Isolation of neural precursor cells from skeletal muscle tissues and their differentiation into neuron-like cells.

    PubMed

    Park, Jung Sik; Kim, Soyeon; Han, Dong Keun; Lee, Ji Youl; Ghil, Sung Ho

    2007-08-31

    Skeletal muscle contains several precursor cells that generate muscle, bone, cartilage and blood cells. Although there are reports that skeletal muscle-derived cells can trans-differentiate into neural-lineage cells, methods for isolating precursor cells, and procedures for successful neural induction have not been fully established. Here, we show that the preplate cell isolation method, which separates cells based on their adhesion characteristics, permits separation of cells possessing neural precursor characteristics from other cells of skeletal muscle tissues. We term these isolated cells skeletal muscle-derived neural precursor cells (SMNPs). The isolated SMNPs constitutively expressed neural stem cell markers. In addition, we describe effective neural induction materials permitting the neuron-like cell differentiation of SMNPs. Treatment with retinoic acid or forskolin facilitated morphological changes in SMNPs; they differentiated into neuron-like cells that possessed specific neuronal markers. These results suggest that the preplate isolation method, and treatment with retinoic acid or forskolin, may provide vital assistance in the use of SMNPs in cell-based therapy of neuronal disease.

  13. Bioinspired magnetite formation from a disordered ferrihydrite-derived precursor.

    PubMed

    Dey, Archan; Lenders, Jos J M; Sommerdijk, Nico A J M

    2015-01-01

    We show that by reacting ferrihydrite (FeH) with Fe((II)) ions and subsequently increasing the pH, magnetite is formed through a multi-step nucleation process mediated by monodisperse FeH-Fe((II)) primary particles. The interaction of these primary particles with a transient green rust phase leads to the formation of smaller secondary particles which form the feedstock for magnetite formation. Surprisingly, the presence of a polypeptide additive prevents the formation of green rust as an Fe((II))-rich intermediate phase, and leads to the formation of amorphous aggregates of FeH-Fe((II)) particles which subsequently transform into the final magnetite nanocrystals. The observation of multiple transitions and the involvement of disordered precursor phases in this bioinspired crystallization route is important for our understanding of the nucleation of magnetite in geological and biological environments, and may lead to new approaches in the sustainable synthesis of this technologically important mineral.

  14. Improved Single-Source Precursors for Solar-Cell Absorbers

    NASA Technical Reports Server (NTRS)

    Banger, Kulbinder K.; Harris, Jerry; Hepp, Aloysius

    2007-01-01

    Improved single-source precursor compounds have been invented for use in spray chemical vapor deposition (spray CVD) of chalcopyrite semiconductor absorber layers of thin-film cells. A "single-source precursor compound" is a single molecular compound that contains all the required elements, which when used under the spray CVD conditions, thermally decomposes to form CuIn(x)Ga(1-x)S(y)Se(2-y).

  15. Skin-derived precursors as a source of progenitors for cutaneous nerve regeneration

    PubMed Central

    Chen, Zhiguo; Pradhan, Sanjay; Liu, Chiachi; Le, Lu Q.

    2012-01-01

    Peripheral nerves have the potential to regenerate axons and reinnervate end organs. Chronic denervation and disturbed nerve regeneration are thought to contribute to peripheral neuropathy, pain and pruritus in the skin. The capacity of denervated distal nerves to support axonal regeneration requires proliferation by Schwann cells, which guide regenerating axons to their denervated targets. However, adult peripheral nerve Schwann cells do not retain a growth-permissive phenotype, as is required to produce new glia. Therefore, it is believed that following injury, mature Schwann cells de-differentiate to a progenitor/stem cell phenotype to promote axonal re-growth. In this study, we show that Skin-derived precursors (SKPs), a recently identified neural-crest related stem cell population in the dermis of skin, are an alternative source of progenitors for cutaneous nerve regeneration. Using in vivo and in vitro 3-D cutaneous nerve regeneration models, we show that the SKPs are neurotropic toward injured nerves and that they have a full capacity to differentiate into Schwann cells and promote axon regeneration. The identification of SKPs as a physiologic source of progenitors for cutaneous nerve regeneration in the skin, where SKPs physiologically reside, has important implications for understanding early cellular events in peripheral nerve regeneration. It also provides fertile ground for the elucidation of intrinsic and extrinsic factors within the nerve microenvironment that likely play essential roles in cutaneous nerve homeostasis. PMID:22851518

  16. Skin-derived precursors as a source of progenitors for cutaneous nerve regeneration.

    PubMed

    Chen, Zhiguo; Pradhan, Sanjay; Liu, Chiachi; Le, Lu Q

    2012-10-01

    Peripheral nerves have the potential to regenerate axons and reinnervate end organs. Chronic denervation and disturbed nerve regeneration are thought to contribute to peripheral neuropathy, pain, and pruritus in the skin. The capacity of denervated distal nerves to support axonal regeneration requires proliferation by Schwann cells, which guide regenerating axons to their denervated targets. However, adult peripheral nerve Schwann cells do not retain a growth-permissive phenotype, as is required to produce new glia. Therefore, it is believed that following injury, mature Schwann cells dedifferentiate to a progenitor/stem cell phenotype to promote axonal regrowth. In this study, we show that skin-derived precursors (SKPs), a recently identified neural crest-related stem cell population in the dermis of skin, are an alternative source of progenitors for cutaneous nerve regeneration. Using in vivo and in vitro three-dimensional cutaneous nerve regeneration models, we show that the SKPs are neurotropic toward injured nerves and that they have a full capacity to differentiate into Schwann cells and promote axon regeneration. The identification of SKPs as a physiologic source of progenitors for cutaneous nerve regeneration in the skin, where SKPs physiologically reside, has important implications for understanding early cellular events in peripheral nerve regeneration. It also provides fertile ground for the elucidation of intrinsic and extrinsic factors within the nerve microenvironment that likely play essential roles in cutaneous nerve homeostasis.

  17. Progranulin promotes the retinal precursor cell proliferation and the photoreceptor differentiation in the mouse retina.

    PubMed

    Kuse, Yoshiki; Tsuruma, Kazuhiro; Sugitani, Sou; Izawa, Hiroshi; Ohno, Yuta; Shimazawa, Masamitsu; Hara, Hideaki

    2016-03-31

    Progranulin (PGRN) is a secreted growth factor associated with embryo development, tissue repair, and inflammation. In a previous study, we showed that adipose-derived stem cell-conditioned medium (ASC-CM) is rich in PGRN. In the present study, we investigated whether PGRN is associated with retinal regeneration in the mammalian retina. We evaluated the effect of ASC-CM using the N-methyl-N-nitrosourea-induced retinal damage model in mice. ASC-CM promoted the differentiation of photoreceptor cells following retinal damage. PGRN increased the number of BrdU(+) cells in the outer nuclear layer following retinal damage some of which were Rx (retinal precursor cell marker) positive. PGRN also increased the number of rhodopsin(+) photoreceptor cells in primary retinal cell cultures. SU11274, a hepatocyte growth factor (HGF) receptor inhibitor, attenuated the increase. These findings suggest that PGRN may affect the differentiation of retinal precursor cells to photoreceptor cells through the HGF receptor signaling pathway.

  18. Differential Effects of Isoxazole-9 on Neural Stem/Progenitor Cells, Oligodendrocyte Precursor Cells, and Endothelial Progenitor Cells

    PubMed Central

    Maki, Takakuni; Shindo, Akihiro; Osumi, Noriko; Zhao, Jing; Lin, Hong; Holder, Julie C.; Chuang, Tsu Tshen; McNeish, John D.; Arai, Ken; Lo, Eng H.

    2015-01-01

    Adult mammalian brain can be plastic after injury and disease. Therefore, boosting endogenous repair mechanisms would be a useful therapeutic approach for neurological disorders. Isoxazole-9 (Isx-9) has been reported to enhance neurogenesis from neural stem/progenitor cells (NSPCs). However, the effects of Isx-9 on other types of progenitor/precursor cells remain mostly unknown. In this study, we investigated the effects of Isx-9 on the three major populations of progenitor/precursor cells in brain: NSPCs, oligodendrocyte precursor cells (OPCs), and endothelial progenitor cells (EPCs). Cultured primary NSPCs, OPCs, or EPCs were treated with various concentrations of Isx-9 (6.25, 12.5, 25, 50 μM), and their cell numbers were counted in a blinded manner. Isx-9 slightly increased the number of NSPCs and effectively induced neuronal differentiation of NSPCs. However, Isx-9 significantly decreased OPC number in a concentration-dependent manner, suggesting cytotoxicity. Isx-9 did not affect EPC cell number. But in a matrigel assay of angiogenesis, Isx-9 significantly inhibited tube formation in outgrowth endothelial cells derived from EPCs. This potential anti-tube-formation effect of Isx-9 was confirmed in a brain endothelial cell line. Taken together, our data suggest that mechanisms and targets for promoting stem/progenitor cells in the central nervous system may significantly differ between cell types. PMID:26407349

  19. Altered Processing of Amyloid Precursor Protein in Cells Undergoing Apoptosis

    PubMed Central

    Fiorelli, Tina; Kirouac, Lisa; Padmanabhan, Jaya

    2013-01-01

    Altered proteolysis of amyloid precursor protein is an important determinant of pathology development in Alzheimer's disease. Here, we describe the detection of two novel fragments of amyloid precursor protein in H4 neuroglioma cells undergoing apoptosis. Immunoreactivity of these 25–35 kDa fragments to two different amyloid precursor protein antibodies suggests that they contain the amyloid-β region and an epitope near the C-terminus of amyloid precursor protein. Generation of these fragments is associated with cleavage of caspase-3 and caspase-7, suggesting activation of these caspases. Studies in neurons undergoing DNA damage-induced apoptosis also showed similar results. Inclusion of caspase inhibitors prevented the generation of these novel fragments, suggesting that they are generated by a caspase-dependent mechanism. Molecular weight prediction and immunoreactivity of the fragments generated suggested that such fragments could not be generated by cleavage at any previously identified caspase, secretase, or calpain site on amyloid precursor protein. Bioinformatic analysis of the amino acid sequence of amyloid precursor protein revealed that fragments fitting the observed size and immunoreactivity could be generated by either cleavage at a novel, hitherto unidentified, caspase site or at a previously identified matrix metalloproteinase site in the extracellular domain. Proteolytic cleavage at any of these sites leads to a decrease in the generation of α-secretase cleaved secreted APP, which has both anti-apoptotic and neuroprotective properties, and thus may contribute to neurodegeneration in Alzheimer's disease. PMID:23469123

  20. Oligodendrocyte Precursor Cells Synthesize Neuromodulatory Factors

    PubMed Central

    Sakry, Dominik; Yigit, Hatice; Dimou, Leda; Trotter, Jacqueline

    2015-01-01

    NG2 protein-expressing oligodendrocyte progenitor cells (OPC) are a persisting and major glial cell population in the adult mammalian brain. Direct synaptic innervation of OPC by neurons throughout the brain together with their ability to sense neuronal network activity raises the question of additional physiological roles of OPC, supplementary to generating myelinating oligodendrocytes. In this study we investigated whether OPC express neuromodulatory factors, typically synthesized by other CNS cell types. Our results show that OPC express two well-characterized neuromodulatory proteins: Prostaglandin D2 synthase (PTGDS) and neuronal Pentraxin 2 (Nptx2/Narp). Expression levels of the enzyme PTGDS are influenced in cultured OPC by the NG2 intracellular region which can be released by cleavage and localizes to glial nuclei upon transfection. Furthermore PTGDS mRNA levels are reduced in OPC from NG2-KO mouse brain compared to WT cells after isolation by cell sorting and direct analysis. These results show that OPC can contribute to the expression of these proteins within the CNS and suggest PTGDS expression as a downstream target of NG2 signaling. PMID:25966014

  1. Comparison of the Transcriptomes of Mouse Skin Derived Precursors (SKPs) and SKP-Derived Fibroblasts (SFBs) by RNA-Seq

    PubMed Central

    Mao, Yujie; Xiong, Lidan; Wang, Siyu; Zhong, Jianqiao; Zhou, Rongying; Li, Li

    2015-01-01

    Skin-derived precursors (SKPs) from dermis possess the capacities of self-renewal and multipotency. In vitro and in vivo studies demonstrated that they can differentiate into fibroblasts. However, little is known about the molecular mechanism of the differentiation of SKPs into fibroblasts. Here we compare the transcriptomes of mouse SKPs and SKP-derived fibroblasts (SFBs) by RNA-Seq analysis, trying to find differences in gene expression between the two kinds of cells and then elucidate the candidate genes that may play important roles in the differentiation of SKPs into fibroblasts. A total of 1971 differentially expressed genes (DEGs) were identified by RNA-Seq, which provided abundant data for further analysis. Gene Ontology enrichment analysis revealed that genes related to cell differentiation, cell proliferation, protein binding, transporter activity and membrane were significantly enriched. The most significantly up-regulated genes Wnt4, Wisp2 and Tsp-1 and down-regulated genes Slitrk1, Klk6, Agtr2, Ivl, Msx1, IL15, Atp6v0d2, Kcne1l and Thbs4 may play important roles in the differentiation of SKPs into fibroblasts. KEGG analysis showed that DEGs were significantly enriched in the TGF-β signaling pathway, Wnt signaling pathway and Notch signaling pathway, which have been previously proven to regulate the differentiation and self-renewal of various stem cells. These identified DEGs and pathways could facilitate further investigations of the detailed molecular mechanisms, making it possible to take advantage of the potential therapeutic applications of SKPs in skin regeneration in the future. PMID:25719759

  2. The endothelin receptor-B is required for the migration of neural crest-derived melanocyte and enteric neuron precursors.

    PubMed

    Lee, Hyung-Ok; Levorse, John M; Shin, Myung K

    2003-07-01

    Mutations in the genes encoding endothelin receptor-B (Ednrb) and its ligand endothelin-3 (Edn3) affect the development of two neural crest-derived cell types, melanocytes and enteric neurons. EDNRB signaling is exclusively required between E10.5 and E12.5 during the migratory phase of melanoblast and enteric neuroblast development. To determine the fate of Ednrb-expressing cells during this critical period, we generated a strain of mice with the bacterial beta-galactosidase (lacZ) gene inserted downstream of the endogenous Ednrb promoter. The expression of the lacZ gene was detected in melanoblasts and precursors of the enteric neuron system (ENS), as well as other neural crest cells and nonneural crest-derived lineages. By comparing Ednrb(lacZ)/+ and Ednrb(lacZ)/Ednrb(lacZ) embryos, we determined that the Ednrb pathway is not required for the initial specification and dispersal of melanoblasts and ENS precursors from the neural crest progenitors. Rather, the EDNRB-mediated signaling is required for the terminal migration of melanoblasts and ENS precursors, and this pathway is not required for the survival of the migratory cells.

  3. Muscle precursor cells invade and repopulate freeze-killed muscles.

    PubMed

    Morgan, J E; Coulton, G R; Partridge, T A

    1987-10-01

    A problem with the use of muscle grafting as a therapeutic procedure is to produce a graft functionally adequate to replace a muscle of complex architecture, such as a sphincter muscle. We thought it might be possible to use dead cadaver muscles, repopulated by the patient's own muscle precursor cells (mpc), to reconstruct muscles whose anatomy would be imposed by the framework of dead muscle and whose genetic constitution would be determined by the mpc. Here we show, in the mouse, that an extensor digitorum longus (EDL) muscle, killed by repeated freezing and thawing, repopulated with mpc and grafted into a nu/nu or tolerant AKR host mouse, is capable of supporting muscle formation. By using the allotypic isoenzyme forms of glucose-6-phosphate isomerase as markers, we have shown that the newly regenerated muscle in such grafts is derived mainly from the implanted mpc, but also to some extent from the host mouse's own mpc. By 50-70 days after grafting, new muscle fibres were found to constitute up to 70% of the graft. Many fibres had assumed diameters in the normal range for mouse muscle, often having peripherally placed nuclei. These findings raise the possibility of the therapeutic use of such grafts. To our surprise, dead EDL muscle grafts into which no mpc had been implanted were also the site of good muscle regeneration. New-formed muscle in these grafts was shown to be derived entirely from mpc which must have migrated into the graft from the host. Investigation of the mechanisms underlying this phenomenon should further our knowledge of factors which regulate the proliferation and movement of dormant mpc in adult animals.

  4. Mesenchymal precursor cells in the blood of normal individuals

    PubMed Central

    Zvaifler, Nathan J; Marinova-Mutafchieva, Lilla; Adams, Gill; Edwards, Christopher J; Moss, Jill; Burger, Jan A; Maini, Ravinder N

    2000-01-01

    collagen, and BMP receptors (heterodimeric structures expressed on mesenchymal lineage cells). The cultured cells also stained strongly for the SH-2 (endoglin) antigen, a putative marker for marrow MSCs. BMPCs express the gene for SDF-1, a potent stroma-derived CXCα chemokine. Discussion: In the circulation of normal individuals is a small population of CD34- mononuclear cells that proliferate rapidly in culture as an adherent population with a variable morphology, display cytoskeletal, cytoplasmic, and surface markers of mesenchymal precursors, and differentiate into several lineages (fibroblasts, osteoblasts, and adipocytes). These are all features found in bone-marrow-derived MSCs. Therefore, autologous blood could provide cells useful for tissue engineering and gene therapy. In addition, the demonstration of similar cells in the inflammatory joint fluids and synovium of patients with rheumatoid arthritis (RA) suggests that these cells may play a role in the pathogenesis of RA. PMID:11056678

  5. A novel treatment for lupus nephritis: lignan precursor derived from flax.

    PubMed

    Clark, W F; Muir, A D; Westcott, N D; Parbtani, A

    2000-01-01

    Flaxseed has renoprotective effects in animal and human lupus nephritis. We have recently extracted the lignan precursor (secoisolariresinol diglucoside) (SDG) to determine if this more palatable derivative of flaxseed would exert renoprotection similar to the whole flaxseed in the aggressive MRL/lpr lupus mouse model. 131 MRL/lpr mice were randomly assigned to saline gavage, 600, 1,200 and 4,800 microg lignan gavage groups. At 7 weeks, 6 animals underwent platelet aggregating factor (PAF) lethal challenge and 40 were studied with urine collection to determine the levels of secoisolariresinol, enterodiol and enterolactone in the gavaged animals. A baseline study of 10 saline gavaged animals took place at 6 weeks. 25 animals in the saline gavage, 600 and 1200 microg lignan groups were studied at 14 and 22 weeks for GFR, spleen lymphocyte S-phase and organ weight studies. Metabolic studies indicated that secoisolariresinol is the major metabolite absorbed and the lowest lignan dose provides a lengthening in survival for the PAF lethal challenge. Body weight, fluid and water intake studies demonstrated that the lignan was well tolerated. Changes in proteinuria, GFR and renal size showed a time- and dose-dependent protection for the lignan precursor. Cervical lymph node size and spleen lymphocyte cells in the S-phase demonstrated modest dose-dependent reductions in the lignan gavaged groups. SDG was converted in the gut to secoisolariresinol, which was absorbed and well tolerated by the MRL/lpr mice. Renoprotection was evidenced, in a dose-dependent fashion, by a significant delay in the onset of proteinuria with preservation in GFR and renal size. This study suggests that SDG may have a therapeutic role in lupus nephritis.

  6. Peripheral Brain Derived Neurotrophic Factor Precursor Regulates Pain as an Inflammatory Mediator

    PubMed Central

    Luo, Cong; Zhong, Xiao-Lin; Zhou, Fiona H.; Li, Jia-yi; Zhou, Pei; Xu, Jun-Mei; Song, Bo; Li, Chang-Qi; Zhou, Xin-Fu; Dai, Ru-Ping

    2016-01-01

    The precursor of brain derived neurotrophic factor (proBDNF), the unprocessed BDNF gene product, binds to its receptors and exerts the opposing biologic functions of mature BDNF. proBDNF is expressed in the peripheral tissues but the functions of peripheral proBDNF remain elusive. Here we showed that proBDNF and its predominant receptor, p75 pan-neurotrophin receptor were upregulated in the nerve fibers and inflammatory cells in the local tissue in inflammatory pain. Neutralization of proBDNF by polyclonal antibody attenuated pain in different models of inflammatory pain. Unilateral intra-plantar supplementation of proBDNF by injecting exogenous proBDNF or ectopic overexpression resulted in pain hypersensitivity and induced spinal phosphorylated extracellular signal-regulated kinase activation. Exogenous proBDNF injection induced the infiltration of inflammatory cells and the activation of proinflammatory cytokines, suggesting that inflammatory reaction contributed to the pro-algesic effect of proBDNF. Finally, we generated monoclonal anti-proBDNF antibody that could biologically block proBDNF. Administration of monoclonal Ab-proBDNF attenuated various types of inflammatory pain and surgical pain. Thus, peripheral proBDNF is a potential pain mediator and anti-proBDNF pretreatment may alleviate the development of inflammatory pain. PMID:27251195

  7. Cerebellar granule cells are predominantly generated by terminal symmetric divisions of granule cell precursors.

    PubMed

    Nakashima, Kie; Umeshima, Hiroki; Kengaku, Mineko

    2015-06-01

    Neurons in the central nervous system (CNS) are generated by symmetric and asymmetric cell division of neural stem cells and their derivative progenitor cells. Cerebellar granule cells are the most abundant neurons in the CNS, and are generated by intensive cell division of granule cell precursors (GCPs) during postnatal development. Dysregulation of GCP cell cycle is causal for some subtypes of medulloblastoma. However, the details and mechanisms underlying neurogenesis from GCPs are not well understood. Using long-term live-cell imaging of proliferating GCPs transfected with a fluorescent newborn-granule cell marker, we found that GCPs underwent predominantly symmetric divisions, generating two GCPs or two neurons, while asymmetric divisions generating a GCP and a neuron were only occasionally observed, in both dissociated culture and within tissues of isolated cerebellar lobules. We found no significant difference in cell cycle length between proliferative and neurogenic divisions, or any consistent changes in cell cycle length during repeated proliferative division. Unlike neural stem cells in the cerebral cortex and spinal cord, which generate many neurons by repeated asymmetric division, cerebellar GCPs produce neurons predominantly by terminal symmetric division. These results indicate diverse mechanisms of neurogenesis in the mammalian brain. © 2015 Wiley Periodicals, Inc.

  8. CD34+ hematopoietic precursors are present in human decidua and differentiate into natural killer cells upon interaction with stromal cells

    PubMed Central

    Vacca, Paola; Vitale, Chiara; Montaldo, Elisa; Conte, Romana; Cantoni, Claudia; Fulcheri, Ezio; Darretta, Valeria; Moretta, Lorenzo; Mingari, Maria Cristina

    2011-01-01

    Natural killer (NK) cells are the main lymphoid population in the maternal decidua during the first trimester of pregnancy. Decidual NK (dNK) cells display a unique functional profile and play a key role in promoting tissue remodeling, neoangiogenesis, and immune modulation. However, little information exists on their origin and development. Here we discovered CD34+ hematopoietic precursors in human decidua (dCD34+). We show that dCD34+ cells differ from cord blood- or peripheral blood-derived CD34+ precursors. The expression of IL-15/IL-2 receptor common β-chain (CD122), IL-7 receptor α-chain (CD127), and mRNA for E4BP4 and ID2 transcription factors suggested that dCD34+ cells are committed to the NK cell lineage. Moreover, they could undergo in vitro differentiation into functional (i.e., IL-8– and IL-22–producing) CD56brightCD16−KIR+/− NK cells in the presence of growth factors or even upon coculture with decidual stromal cells. Their NK cell commitment was further supported by the failure to undergo myeloid differentiation in the presence of GM-CSF. Our findings strongly suggest that decidual NK cells may directly derive from CD34+ cell precursors present in the decidua upon specific cellular interactions with components of the decidual microenvironment. PMID:21248224

  9. Skin-Derived Precursors against UVB-Induced Apoptosis via Bcl-2 and Nrf2 Upregulation

    PubMed Central

    Zhong, Jianqiao

    2016-01-01

    Bcl-2 and Nrf2 are critical factors in protecting cells against UVB-induced apoptosis. Hair-follicle-bulge stem cells could resist ionization through Bcl-2 upregulation. Skin-derived precursors (SKPs) dwelling on the bulge may be against UVB irradiation. Initially, SKPs were isolated and identified. Then, SKPs were exposed to UVB and grew in medium for 24 hours. CCK-8 assay, TUNEL, and Ki67 staining evaluated cells apoptosis/proliferation, while SA-βgal staining evaluated cells senescence and pH2AX immunostaining evaluated DNA damage. Meanwhile, Bcl-2, Nrf2, HO-1, Bax, and Bak expressions were assessed by qRT-PCR and western blot. Two weeks later, floating spheres appeared and were identified as SKPs. After UVB radiation, SKPs maintained spherical colonies and outnumbered unirradiated ones, showing high Ki67 expression and low TUNEL, SA-βgal, and pH2AX expression. Fibroblasts (FBs), however, displayed deformation, senescence, and reduction, with increased TUNEL, SA-βgal, and pH2AX expression. Moreover, Bcl-2 and Nrf2 mRNA expression were significantly higher than Bak and Bax in irradiated SKPs. Conversely, Bcl-2 and Nrf2 mRNA levels greatly decreased compared with Bax and Bak in irradiated FBs. Interestingly, SKPs showed higher protein levels of Bcl-2, Nrf2, and HO-1 than FBs. SKPs exert a beneficial effect on resisting UVB-induced apoptosis, which may be associated with Bcl-2 and Nrf2 upregulation. PMID:27635399

  10. Glial-restricted precursors as potential candidates for ALS cell-replacement therapy.

    PubMed

    Kruminis-Kaszkiel, Ewa; Wojtkiewicz, Joanna; Maksymowicz, Wojciech

    2014-01-01

    Amyotrophic lateral sclerosis is a multifactorial progressive neurodegenerative disorder leading to severe disability and death within 3-5 years after diagnosis. The main mechanisms underlying the disease progression are poorly known but according to the current knowledge, neuroinflammation is a key player in motor neurons damage. Astrocytes constitute an important cell population involved in neuroinflammatory reaction. Many studies confirmed their striking connection with motor neuron pathology and therefore they might be a target for the treatment of ALS. Cell-based therapy appears to be a promising strategy. Since direct replacement or restoring of motor neurons using various stem cells is challenging, enrichment of healthy donor-derived astrocytes appears to be a more realistic and beneficial approach. The effects of astrocytes have been examined using transplantation of glial-restricted precursors (GRPs) that represent one of the earliest precursors within the oligodendrocytic and astrocytic cell lineage. In this review, we focused on evidence-based data on astrocyte replacement transplantation therapy using GRPs in animal models of motor neuron diseases. The efficacy of GRPs engrafting is very encouraging. Furthermore, the lesson learned from application of lineage-restricted precursors in spinal cord injury (SCI) indicates that differentiation of GRPs into astrocytes before transplantation might be more advantageous in the context of axon regeneration. To sum up, the studies of glial-restricted precursors have made a step forward to ALS research and might bring breakthroughs to the field of ALS therapy in the future.

  11. Ricin and Ricinus communis agglutinin subunits are all derived from a single-size polypeptide precursor.

    PubMed

    Butterworth, A G; Lord, J M

    1983-12-01

    Antibodies have been raised in rabbits against the individually purified A and B subunits of the toxic castor bean lectin, ricin, and against the A' and B' subunits of Ricinus communis agglutinin type I. Each of the antisera recognised a single polypeptide species of Mr 60 500 when maturing castor bean endosperm mRNA was translated in vitro in a rabbit-reticulocyte-derived system. When dog pancreatic microsomal vesicles were included in the translational system, each subunit antiserum precipitated a group of 66 000-68 000-Mr core-glycosylated polypeptides which had been translocated into the lumen of the vesicles. The 60 500-Mr polypeptide appeared to be a common precursor to all four individual lectin subunits since (a) its glycosylated (66 000-68 000-Mr) forms were readily detected in the endoplasmic reticulum fraction isolated from maturing castor bean endosperm and (b) pulse-chase studies showed that the glycosylated precursors disappeared from the endoplasmic reticulum fraction with the concomittant appearance of authentic lectin subunits in a soluble protein fraction which included protein body matrix components. Antiserum prepared against whole R. communis agglutinin, type I, also precipitated the 65 000-Mr precursor in vitro and in vivo, but in addition precipitated a non-glycosylated 34 000-Mr polypeptide. This smaller protein is not a lectin subunit precursor, contradicting an earlier suggestion. It is most probably a precursor to the 2-S albumin storage proteins found in castor bean endosperm protein bodies.

  12. Thin film solar cells by selenization sulfurization using diethyl selenium as a selenium precursor

    SciTech Connect

    Dhere, Neelkanth G.; Kadam, Ankur A.

    2009-12-15

    A method of forming a CIGSS absorber layer includes the steps of providing a metal precursor, and selenizing the metal precursor using diethyl selenium to form a selenized metal precursor layer (CIGSS absorber layer). A high efficiency solar cell includes a CIGSS absorber layer formed by a process including selenizing a metal precursor using diethyl selenium to form the CIGSS absorber layer.

  13. 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.

  14. 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

  15. Suiciding of lymphocytic precursor cells by tritiated nucleosides, in vitro.

    PubMed

    Uyeki, E M; Nishimura, T; Bisel, T U

    1978-02-01

    Differences in suiciding by various tritiated nucleosides were observed between two functional assays for in vitro lymphocytic precursor cell development, the hemolysin plaque-forming cell (PFC) assay and the B lymphocytic colony-forming cell (CFC-L) assay, using BDF1 mouse spleen cells. PFC growth was markedly reduced by an early (days 0-1) pulse of tritiated deoxyadenosine ([3H]dAdo), but relatively unaffected by a pulse of tritiated thymidine ([3H]dThd) during the same interval. In contrast, CFC-L formation significantly dropped after an early (day 0) [3H]dThd pulse, as well as after pulses of [3H]dAdo and the corresponding tritiated ribosides, uridine and adenosine. This implied a cycling state in an early lymphocytic precursor cell, as opposed to the PFC insensitivity to an early [3H]dThd pulse. The response pattern of colonies and clusters to [3H]dThd supported our notion of a delayed suiciding of CFC contributing to the increase in cluster numbers.

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

    PubMed Central

    Sauter, H; Paige, C J

    1987-01-01

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

  17. Highly efficient differentiation of neural precursors from human embryonic stem cells and benefits of transplantation after ischemic stroke in mice.

    PubMed

    Drury-Stewart, Danielle; Song, Mingke; Mohamad, Osama; Guo, Ying; Gu, Xiaohuan; Chen, Dongdong; Wei, Ling

    2013-08-08

    Ischemic stroke is a leading cause of death and disability, but treatment options are severely limited. Cell therapy offers an attractive strategy for regenerating lost tissues and enhancing the endogenous healing process. In this study, we investigated the use of human embryonic stem cell-derived neural precursors as a cell therapy in a murine stroke model. Neural precursors were derived from human embryonic stem cells by using a fully adherent SMAD inhibition protocol employing small molecules. The efficiency of neural induction and the ability of these cells to further differentiate into neurons were assessed by using immunocytochemistry. Whole-cell patch-clamp recording was used to demonstrate the electrophysiological activity of human embryonic stem cell-derived neurons. Neural precursors were transplanted into the core and penumbra regions of a focal ischemic stroke in the barrel cortex of mice. Animals received injections of bromodeoxyuridine to track regeneration. Neural differentiation of the transplanted cells and regenerative markers were measured by using immunohistochemistry. The adhesive removal test was used to determine functional improvement after stroke and intervention. After 11 days of neural induction by using the small-molecule protocol, over 95% of human embryonic stem-derived cells expressed at least one neural marker. Further in vitro differentiation yielded cells that stained for mature neuronal markers and exhibited high-amplitude, repetitive action potentials in response to depolarization. Neuronal differentiation also occurred after transplantation into the ischemic cortex. A greater level of bromodeoxyuridine co-localization with neurons was observed in the penumbra region of animals receiving cell transplantation. Transplantation also improved sensory recovery in transplant animals over that in control animals. Human embryonic stem cell-derived neural precursors derived by using a highly efficient small-molecule SMAD inhibition

  18. Epigenetic mechanisms regulating differentiation of neural stem/precursor cells.

    PubMed

    Adefuin, Aliya Mari D; Kimura, Ayaka; Noguchi, Hirofumi; Nakashima, Kinichi; Namihira, Masakazu

    2014-01-01

    Differentiation of neural stem/precursor cells (NS/PCs) into neurons, astrocytes and oligodendrocytes during mammalian brain development is a carefully controlled and timed event. Increasing evidences suggest that epigenetic regulation is necessary to drive this. Here, we provide an overview of the epigenetic mechanisms involved in the developing mammalian embryonic forebrain. Histone methylation is a key factor but other epigenetic factors such as DNA methylation and noncoding RNAs also partake during fate determination. As numerous epigenetic modifications have been identified, future studies on timing and regional specificity of these modifications will further deepen our understanding of how intrinsic and extrinsic mechanisms participate together to precisely control brain development.

  19. Oligodendrocyte precursor cells generate pituicytes in vivo during neurohypophysis development.

    PubMed

    Virard, Isabelle; Coquillat, Delphine; Bancila, Mircea; Kaing, Sovann; Durbec, Pascale

    2006-02-01

    In the vertebrate brain, much remains to be understood concerning the origin of glial cell diversity and the potential lineage relationships between the various types of glia. Besides astrocytes and myelin-forming oligodendrocytes, other macroglial cell populations are found in discrete areas of the central nervous system (CNS). They share functional features with astrocytes and oligodendrocytes but also display specific characteristics. Such specialized cells, called pituicytes, are located in the neurohypophysis (NH). Our work focuses on the lineage of the pituicytes during rodent development. First, we show that cells identified with a combination of oligodendrocyte precursor cell (OPC) markers are present in the developing rat NH. In culture, neonatal NH progenitors also share major functional characteristics with OPCs, being both migratory and bipotential, i.e. able to give rise to type 2 astrocytes and oligodendrocytes. We then observe that, either in vitro or after transplantation into myelin-deficient Shiverer brain, pieces of NH generate myelinating oligodendrocytes, confirming the oligodendrogenic potentiality of NH cells. However, no mature oligodendrocyte can be found in the NH. This led us to hypothesize that the OPCs present in the developing NH might be generating other glial cells, especially the pituicytes. Consistent with this hypothesis, the OPCs appear during NH development before pituicytes differentiate. Finally, we establish a lineage relationship between olig1+ cells, most likely OPCs, and the pituicytes by fate-mapping experiments using genetically engineered mice. This constitutes the first demonstration that OPCs generate glial cells other than oligodendrocytes in vivo.

  20. Ebf2 is a selective marker of brown and beige adipogenic precursor cells.

    PubMed

    Wang, Wenshan; Kissig, Megan; Rajakumari, Sona; Huang, Li; Lim, Hee-Woong; Won, Kyoung-Jae; Seale, Patrick

    2014-10-07

    Brown adipocytes and muscle and dorsal dermis descend from precursor cells in the dermomyotome, but the factors that regulate commitment to the brown adipose lineage are unknown. Here, we prospectively isolated and determined the molecular profile of embryonic brown preadipose cells. Brown adipogenic precursor activity in embryos was confined to platelet-derived growth factor α(+), myogenic factor 5(Cre)-lineage-marked cells. RNA-sequence analysis identified early B-cell factor 2 (Ebf2) as one of the most selectively expressed genes in this cell fraction. Importantly, Ebf2-expressing cells purified from Ebf2(GFP) embryos or brown fat tissue did not express myoblast or dermal cell markers and uniformly differentiated into brown adipocytes. Interestingly, Ebf2-expressing cells from white fat tissue in adult animals differentiated into brown-like (or beige) adipocytes. Loss of Ebf2 in brown preadipose cells reduced the expression levels of brown preadipose-signature genes, whereas ectopic Ebf2 expression in myoblasts activated brown preadipose-specific genes. Altogether, these results indicate that Ebf2 specifically marks and regulates the molecular profile of brown preadipose cells.

  1. Single Source Precursors for Thin Film Solar Cells

    NASA Technical Reports Server (NTRS)

    Banger, Kulbinder K.; Hollingsworth, Jennifer A.; Harris, Jerry D.; Cowen, Jonathan; Buhro, William E.; Hepp, Aloysius F.

    2002-01-01

    The development of thin film solar cells on flexible, lightweight, space-qualified substrates provides an attractive cost solution to fabricating solar arrays with high specific power, (W/kg). The use of a polycrystalline chalcopyrite absorber layer for thin film solar cells is considered as the next generation photovoltaic devices. At NASA GRC we have focused on the development of new single source precursors (SSP) and their utility to deposit the chalcopyrite semi-conducting layer (CIS) onto flexible substrates for solar cell fabrication. The syntheses and thermal modulation of SSPs via molecular engineering is described. Thin-film fabrication studies demonstrate the SSPs can be used in a spray CVD (chemical vapor deposition) process, for depositing CIS at reduced temperatures, which display good electrical properties, suitable for PV (photovoltaic) devices.

  2. Electronic properties and microstructures of amorphous silicon carbonitrides ceramics derived from polymer precursors

    NASA Astrophysics Data System (ADS)

    Jiang, Tao

    2009-12-01

    Polymer-derived ceramics (PDCs) are a new class of high-temperature materials synthesized by thermal decomposition of polymeric precursors. These materials possess many unique features as compared with conventional ceramics synthesized by powder metallurgy based processing. For example, PDCs are neither amorphous nor crystalline. Instead, they possess nano-domain structures. Due to the direct chemical-to-ceramic processing, PDCs can be used for making components and devices with complex shapes. Thus, understanding the properties and structures of these materials are of both fundamental and practical interest. In this work, the structures and electronic behavior of polymer-derived amorphous silicon carbonitrides (SiCNs) were investigated. The materials were synthesized by pyrolysis of a commercially available liquid precursor. Ceramic materials with varied structures/properties were successfully synthesized by modifying the precursor and using different pyrolysis temperatures. The structures of the obtained materials were studied using XRD, solid state NMR, EPR, FTIR and Raman Spectroscope. The electronic behavior of the materials was investigated by measuring I-V curves, Hall effects, temperature dependent conductivity. The experiments were also performed to measure UV-Visible absorption and dielectric properties of the materials. This work leads to the following significant progresses: (i) developed quantitative technique for measuring free carbon concentration; (ii) achieved better understanding of the electronic conduction mechanisms and measured electronic structures of the materials for the first time; and (iii) demonstrated that these materials possess unusual dielectric behavior and provide qualitative explanations.

  3. Functional analysis of the defective T cell regulation of the antigen-specific PFC response in SLE patients: differentiation of suppressor precursor cells to suppressor effector cells.

    PubMed Central

    Heijnen, C J; Pot, K H; Kater, L; Kluin-Nelemans, H C; Uytdehaag, F; Ballieux, R E

    1982-01-01

    The investigation described here is concerned with the T cell regulation of the antigen-specific antibody response which has been studied in patients suffering from systemic lupus erythematosus (SLE). Apart from the fact that T helper cell activity was found to be less efficient, it appeared that the peripheral blood leucocytes (PBL) of patients in an active stage of the disease did not contain the suppressor precursor cells, which functions as the target cell for the inductive signal of T mu+ suppressor inducer cells. The absence of the suppressor precursor cells in SLE patients coincided with the absence of T gamma+ suppressor effector cells. Characterization of the (post-thymic) precursor cells (derived from normal donors) with the aid of monoclonal antibodies of the OKT series and several other markers pointed out that this population contains OKT4+ as well as OKT8+ cells. Further experiments demonstrated that the cells are capable of rosetting with autologous erythrocytes, and do not bear Fc receptors for IgM or IgG. Considering the various findings as a whole the conclusion is warranted that the post-thymic suppressor precursor T cell can differentiate into a suppressor effector cell only after interaction with T suppressor inducer cells. PMID:6210474

  4. Notch signaling acts before cell division to promote asymmetric cleavage and cell fate of neural precursor cells.

    PubMed

    Bhat, Krishna Moorthi

    2014-10-21

    Asymmetric cell divisions in the central nervous system generate neurons of diverse fates. In Drosophila melanogaster, the protein Numb localizes asymmetrically to dividing neural precursor cells such that only one daughter cell inherits Numb. Numb inhibits Notch signaling in this daughter cell, resulting in a different cell fate from the Notch-induced fate in the other-Numb-negative-daughter cell. Precursor cells undergo asymmetric cytokinesis generating daughter cells of different sizes. I found that inactivation of Notch in fly embryonic neural precursor cells disrupted the asymmetric positioning of the cleavage furrow and produced daughter cells of the same size and fate. Moreover, inactivation of Notch at different times altered the degree of asymmetric Numb localization, such that earlier inactivation of Notch caused symmetric distribution of Numb and later inactivation produced incomplete asymmetric localization of Numb. The extent of asymmetrically localized Numb positively correlated with the degree of asymmetric cytokinesis and the size disparity in daughter cells. Loss of Numb or expression of constitutively active Notch led to premature specification of the precursor cells into the fate of one of the daughter cells. Thus, in addition to its role in the specification of daughter cell fate after division, Notch controls Numb localization in the precursor cells to determine the size and fate of daughter cells. Numb also inhibits Notch signaling in precursor cells to prevent Notch-induced differentiation of the precursor cell, forming an autoregulatory loop.

  5. Brain-derived neurotrophic factor promotes vasculature-associated migration of neuronal precursors toward the ischemic striatum.

    PubMed

    Grade, Sofia; Weng, Yuan C; Snapyan, Marina; Kriz, Jasna; Malva, João O; Saghatelyan, Armen

    2013-01-01

    Stroke induces the recruitment of neuronal precursors from the subventricular zone (SVZ) into the ischemic striatum. In injured areas, de-routed neuroblasts use blood vessels as a physical scaffold to their migration, in a process that resembles the constitutive migration seen in the rostral migratory stream (RMS). The molecular mechanism underlying injury-induced vasculature-mediated migration of neuroblasts in the post-stroke striatum remains, however, elusive. Using adult mice we now demonstrate that endothelial cells in the ischemic striatum produce brain-derived neurotrophic factor (BDNF), a neurotrophin that promotes the vasculature-mediated migration of neuronal precursors in the RMS, and that recruited neuroblasts maintain expression of p75NTR, a low-affinity receptor for BDNF. Reactive astrocytes, which are widespread throughout the damaged area, ensheath blood vessels and express TrkB, a high-affinity receptor for BDNF. Despite the absence of BDNF mRNA, we observed strong BDNF immunolabeling in astrocytes, suggesting that these glial cells trap extracellular BDNF. Importantly, this pattern of expression is reminiscent of the adult RMS, where TrkB-expressing astrocytes bind and sequester vasculature-derived BDNF, leading to the entry of migrating cells into the stationary phase. Real-time imaging of cell migration in acute brain slices revealed a direct role for BDNF in promoting the migration of neuroblasts to ischemic areas. We also demonstrated that cells migrating in the ischemic striatum display higher exploratory behavior and longer stationary periods than cells migrating in the RMS. Our findings suggest that the mechanisms involved in the injury-induced vasculature-mediated migration of neuroblasts recapitulate, at least partially, those observed during constitutive migration in the RMS.

  6. Adult subependymal neural precursors, but not differentiated cells, undergo rapid cathodal migration in the presence of direct current electric fields.

    PubMed

    Babona-Pilipos, Robart; Droujinine, Ilia A; Popovic, Milos R; Morshead, Cindi M

    2011-01-01

    The existence of neural stem and progenitor cells (together termed neural precursor cells) in the adult mammalian brain has sparked great interest in utilizing these cells for regenerative medicine strategies. Endogenous neural precursors within the adult forebrain subependyma can be activated following injury, resulting in their proliferation and migration toward lesion sites where they differentiate into neural cells. The administration of growth factors and immunomodulatory agents following injury augments this activation and has been shown to result in behavioural functional recovery following stroke. With the goal of enhancing neural precursor migration to facilitate the repair process we report that externally applied direct current electric fields induce rapid and directed cathodal migration of pure populations of undifferentiated adult subependyma-derived neural precursors. Using time-lapse imaging microscopy in vitro we performed an extensive single-cell kinematic analysis demonstrating that this galvanotactic phenomenon is a feature of undifferentiated precursors, and not differentiated phenotypes. Moreover, we have shown that the migratory response of the neural precursors is a direct effect of the electric field and not due to chemotactic gradients. We also identified that epidermal growth factor receptor (EGFR) signaling plays a role in the galvanotactic response as blocking EGFR significantly attenuates the migratory behaviour. These findings suggest direct current electric fields may be implemented in endogenous repair paradigms to promote migration and tissue repair following neurotrauma.

  7. The chemokine receptor CXCR6 contributes to recruitment of bone marrow-derived fibroblast precursors in renal fibrosis.

    PubMed

    Xia, Yunfeng; Yan, Jingyin; Jin, Xiaogao; Entman, Mark L; Wang, Yanlin

    2014-08-01

    Bone marrow-derived fibroblasts in circulation are of hematopoietic origin, and they proliferate, differentiate into myofibroblasts, and express the chemokine receptor CXCR6. As chemokines mediate the trafficking of circulating cells to sites of injury, we studied the role of CXCR6 in mouse models of renal injury. Significantly, the kidney of CXCR6 knockout mice accumulated fewer bone marrow-derived fibroblasts in response to injury, expressed less profibrotic chemokines and cytokines, displayed fewer myofibroblasts, and expressed less α-smooth muscle actin in the obstructed kidneys compared with wild-type (WT) mice. CXCR6 deficiency inhibited total collagen deposition and suppressed the expression of collagen I and fibronectin in the obstructed kidneys. Furthermore, WT mice engrafted with CXCR6(-/-) bone marrow cells displayed fewer bone marrow-derived fibroblasts in the kidneys with obstructive injury and showed less severe renal fibrosis compared with WT mice engrafted with CXCR6(+/+) bone marrow cells. Transplant of WT bone marrow into CXCR6(-/-) recipients restored recruitment of myeloid fibroblasts and susceptibility to fibrosis. Hematopoietic fibroblasts migrate into injured kidney and proliferate and differentiate into myofibroblasts. Thus, CXCR6, together with other chemokines and their receptors, may have important roles in the recruitment of bone marrow-derived fibroblast precursors into the kidney and contribute to the pathogenesis of renal fibrosis.

  8. Chemokines influence the migration and fate of neural precursor cells from the young adult and middle-aged rat subventricular zone.

    PubMed

    Gordon, R J; Mehrabi, N F; Maucksch, C; Connor, B

    2012-01-01

    We have previously demonstrated a role for the chemokines MCP-1, MIP-1α and GRO-α in directing subventricular zone (SVZ)-derived neural precursor cell migration towards the site of cell death in the adult rodent brain. However the influence of chemokines such as MCP-1, MIP-1α and GRO-α on the differentiation of adult neural precursor cells has not previously been investigated. Further, as the majority of neurological disorders and injuries occur during ageing, it is important to investigate the effect of chemokines on adult neural precursor cell cultures obtained from the ageing brain. This study therefore examined the effect of MCP-1, MIP-1α and GRO-α on SVZ-derived neural precursor cell differentiation in vitro, and assessed whether precursor cells from the middle-aged rat brain (13 months old) follow the same migratory and differential profile as neural precursor cells obtained from the young adult rat brain (2 months old). We observed that each of the chemokines examined generated differing effects in regards to neuronal or glial differentiation. Further, both MIP-1α and GRO-α increased total cell number, suggesting an effect on precursor cell proliferation and/or survival. In agreement with cultures obtained from young adult brains, SVZ-derived neural precursor cells cultured from the middle-aged brain exhibited chemotactic migration in response to a concentration gradient. These results indicate that the chemokines MCP-1, MIP-1α and GRO-α can influence both the migration and fate choice of SVZ-derived neural precursor cells, as well as promoting cell viability. While a response to each of these chemokines is maintained in the middle-aged brain, a distinct age-related alteration in differential fate can be identified.

  9. Progranulin promotes the retinal precursor cell proliferation and the photoreceptor differentiation in the mouse retina

    PubMed Central

    Kuse, Yoshiki; Tsuruma, Kazuhiro; Sugitani, Sou; Izawa, Hiroshi; Ohno, Yuta; Shimazawa, Masamitsu; Hara, Hideaki

    2016-01-01

    Progranulin (PGRN) is a secreted growth factor associated with embryo development, tissue repair, and inflammation. In a previous study, we showed that adipose-derived stem cell-conditioned medium (ASC-CM) is rich in PGRN. In the present study, we investigated whether PGRN is associated with retinal regeneration in the mammalian retina. We evaluated the effect of ASC-CM using the N-methyl-N-nitrosourea-induced retinal damage model in mice. ASC-CM promoted the differentiation of photoreceptor cells following retinal damage. PGRN increased the number of BrdU+ cells in the outer nuclear layer following retinal damage some of which were Rx (retinal precursor cell marker) positive. PGRN also increased the number of rhodopsin+ photoreceptor cells in primary retinal cell cultures. SU11274, a hepatocyte growth factor (HGF) receptor inhibitor, attenuated the increase. These findings suggest that PGRN may affect the differentiation of retinal precursor cells to photoreceptor cells through the HGF receptor signaling pathway. PMID:27030285

  10. Rejuvenation of MPTP-induced human neural precursor cell senescence by activating autophagy

    SciTech Connect

    Zhu, Liang; Dong, Chuanming; Sun, Chenxi; Ma, Rongjie; Yang, Danjing; Zhu, Hongwen; Xu, Jun

    2015-08-21

    Aging of neural stem cell, which can affect brain homeostasis, may be caused by many cellular mechanisms. Autophagy dysfunction was found in aged and neurodegenerative brains. However, little is known about the relationship between autophagy and human neural stem cell (hNSC) aging. The present study used 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) to treat neural precursor cells (NPCs) derived from human embryonic stem cell (hESC) line H9 and investigate related molecular mechanisms involved in this process. MPTP-treated NPCs were found to undergo premature senescence [determined by increased senescence-associated-β-galactosidase (SA-β-gal) activity, elevated intracellular reactive oxygen species level, and decreased proliferation] and were associated with impaired autophagy. Additionally, the cellular senescence phenotypes were manifested at the molecular level by a significant increase in p21 and p53 expression, a decrease in SOD2 expression, and a decrease in expression of some key autophagy-related genes such as Atg5, Atg7, Atg12, and Beclin 1. Furthermore, we found that the senescence-like phenotype of MPTP-treated hNPCs was rejuvenated through treatment with a well-known autophagy enhancer rapamycin, which was blocked by suppression of essential autophagy gene Beclin 1. Taken together, these findings reveal the critical role of autophagy in the process of hNSC aging, and this process can be reversed by activating autophagy. - Highlights: • We successfully establish hESC-derived neural precursor cells. • MPTP treatment induced senescence-like state in hESC-derived NPCs. • MPTP treatment induced impaired autophagy of hESC-derived NPCs. • MPTP-induced hESC-derived NPC senescence was rejuvenated by activating autophagy.

  11. Extracellular Vesicles from Vascular Endothelial Cells Promote Survival, Proliferation and Motility of Oligodendrocyte Precursor Cells

    PubMed Central

    Kurachi, Masashi; Mikuni, Masahiko; Ishizaki, Yasuki

    2016-01-01

    We previously examined the effect of brain microvascular endothelial cell (MVEC) transplantation on rat white matter infarction, and found that MVEC transplantation promoted remyelination of demyelinated axons in the infarct region and reduced apoptotic death of oligodendrocyte precursor cells (OPCs). We also found that the conditioned medium (CM) from cultured MVECs inhibited apoptosis of cultured OPCs. In this study, we examined contribution of extracellular vesicles (EVs) contained in the CM to its inhibitory effect on OPC apoptosis. Removal of EVs from the CM by ultracentrifugation reduced its inhibitory effect on OPC apoptosis. To confirm whether EVs derived from MVECs are taken up by cultured OPCs, we labeled EVs with PKH67, a fluorescent dye, and added them to OPC cultures. Many vesicular structures labeled with PKH67 were found within OPCs immediately after their addition. Next we examined the effect of MVEC-derived EVs on OPC behaviors. After 2 days in culture with EVs, there was significantly less pyknotic and more BrdU-positive OPCs when compared to control. We also examined the effect of EVs on motility of OPCs. OPCs migrated longer in the presence of EVs when compared to control. To examine whether these effects on cultured OPCs are shared by EVs from endothelial cells, we prepared EVs from conditioned media of several types of endothelial cells, and tested their effects on cultured OPCs. EVs from all types of endothelial cells we examined reduced apoptosis of OPCs and promoted their motility. Identification of the molecules contained in EVs from endothelial cells may prove helpful for establishment of effective therapies for demyelinating diseases. PMID:27403742

  12. Syntheses of biodiesel precursors: sulfonic acid catalysts for condensation of biomass-derived platform molecules.

    PubMed

    Balakrishnan, Madhesan; Sacia, Eric R; Bell, Alexis T

    2014-04-01

    Synthesis of transportation fuel from lignocellulosic biomass is an attractive solution to the green alternative-energy problem. The production of biodiesel, in particular, involves the process of upgrading biomass-derived small molecules to diesel precursors containing a specific carbon range (C11 -C23). Herein, a carbon-upgrading process utilizing an acid-catalyzed condensation of furanic platform molecules from biomass is described. Various types of sulfonic acid catalysts have been evaluated for this process, including biphasic and solid supported catalysts. A silica-bound alkyl sulfonic acid catalyst has been developed for promoting carbon-carbon bond formation of biomass-derived carbonyl compounds with 2-methylfuran. This hydrophobic solid acid catalyst exhibits activity and selectivity that are comparable to those of a soluble acid catalyst. The catalyst can be readily recovered and recycled, possesses appreciable hydrolytic stability in the presence of water, and retains its acidity over multiple reaction cycles. Application of this catalyst to biomass-derived platform molecules led to the synthesis of a variety of furanic compounds, which are potential biodiesel precursors.

  13. Myeloid-derived suppressor cells in gliomas

    PubMed Central

    Kaminska, Bozena

    2016-01-01

    Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of early myeloid progenitors and precursors at different stages of differentiation into granulocytes, macrophages, and dendritic cells. Blockade of their differentiation into mature myeloid cells in cancer results in an expansion of this population. High-grade gliomas are the most common malignant tumours of the central nervous system (CNS), with a poor prognosis despite intensive radiation and chemotherapy. Histopathological and flow cytometry analyses of human and rodent experimental gliomas revealed the extensive heterogeneity of immune cells infiltrating gliomas and their microenvironment. Immune cell infiltrates consist of: resident (microglia) and peripheral macrophages, granulocytes, myeloid-derived suppressor cells, and T lymphocytes. Intratumoural density of glioma-associated MDSCs correlates positively with the histological grade of gliomas and patient’s survival. MDSCs have the ability to attract T regulatory lymphocytes to the tumour, but block the activation of tumour-reactive CD4+ T helper cells and cytotoxic CD8+ T cells. Immunomodulatory mechanisms employed by malignant gliomas pose an appalling challenge to brain tumour immunotherapy. In this mini-review we describe phenotypic and functional characteristics of MDSCs in humans and rodents, and their occurrence and potential roles in glioma progression. While understanding the complexity of immune cell interactions in the glioma microenvironment is far from being accomplished, there is significant progress that may lead to the development of immunotherapy for gliomas. PMID:28373814

  14. Neural stem cells isolated from amyloid precursor protein-mutated mice for drug discovery.

    PubMed

    Baldassarro, Vito Antonio; Lizzo, Giulia; Paradisi, Michela; Fernández, Mercedes; Giardino, Luciana; Calzà, Laura

    2013-10-26

    To develop an in vitro model based on neural stem cells derived from transgenic animals, to be used in the study of pathological mechanisms of Alzheimer's disease and for testing new molecules. Neural stem cells (NSCs) were isolated from the subventricular zone of Wild type (Wt) and Tg2576 mice. Primary and secondary neurosphere generation was studied, analysing population doubling and the cell yield per animal. Secondary neurospheres were dissociated and plated on MCM Gel Cultrex 2D and after 6 d in vitro (DIVs) in mitogen withdrawal conditions, spontaneous differentiation was studied using specific neural markers (MAP2 and TuJ-1 for neurons, GFAP for astroglial cells and CNPase for oligodendrocytes). Gene expression pathways were analysed in secondary neurospheres, using the QIAGEN PCR array for neurogenesis, comparing the Tg2576 derived cell expression with the Wt cells. Proteins encoded by the altered genes were clustered using STRING web software. As revealed by 6E10 positive staining, all Tg2576 derived cells retain the expression of the human transgenic Amyloid Precursor Protein. Tg2576 derived primary neurospheres show a decrease in population doubling. Morphological analysis of differentiated NSCs reveals a decrease in MAP2- and an increase in GFAP-positive cells in Tg2576 derived cells. Analysing the branching of TuJ-1 positive cells, a clear decrease in neurite number and length is observed in Tg2576 cells. The gene expression neurogenesis pathway revealed 11 altered genes in Tg2576 NSCs compared to Wt. Tg2576 NSCs represent an appropriate AD in vitro model resembling some cellular alterations observed in vivo, both as stem and differentiated cells.

  15. Acetylcholine stimulates cortical precursor cell proliferation in vitro via muscarinic receptor activation and MAP kinase phosphorylation.

    PubMed

    Ma, W; Maric, D; Li, B S; Hu, Q; Andreadis, J D; Grant, G M; Liu, Q Y; Shaffer, K M; Chang, Y H; Zhang, L; Pancrazio, J J; Pant, H C; Stenger, D A; Barker, J L

    2000-04-01

    Increasing evidence has shown that some neurotransmitters act as growth-regulatory signals during brain development. Here we report a role for the classical neurotransmitter acetylcholine (ACh) to stimulate proliferation of neural stem cells and stem cell-derived progenitor cells during neural cell lineage progression in vitro. Neuroepithelial cells in the ventricular zone of the embryonic rat cortex were found to express the m2 subtype of the muscarinic receptor. Neural precursor cells dissociated from the embryonic rat cortical neuroepithelium were expanded in culture with basic fibroblast growth factor (bFGF). reverse transcriptase-polymerase chain reaction (RT-PCR) revealed the presence of m2, m3 and m4 muscarinic receptor subtype transcripts, while immunocytochemistry demonstrated m2 protein. ACh and carbachol induced an increase in cytosolic Ca2+ and membrane currents in proliferating (BrdU+) cells, both of which were abolished by atropine. Exposure of bFGF-deprived precursor cells to muscarinic agonists not only increased both cell number and DNA synthesis, but also enhanced differentiation of neurons. These effects were blocked by atropine, indicating the involvement of muscarinic ACh receptors. The growth-stimulating effects were also antagonized by a panel of inhibitors of second messengers, including 1,2-bis-(O-aminophenoxy)-ethane-N,N,N', N'-tetraacetic acid (BAPTA-AM) to chelate cytosolic Ca2+, EGTA to complex extracellular Ca2+, pertussis toxin, which uncouples certain G-proteins, the protein kinase C inhibitor H7 and the mitogen-activated protein kinase (MAPK) inhibitor PD98059. Muscarinic agonists activated MAPK, which was significantly inhibited by atropine and the same panel of inhibitors. Thus, muscarinic receptors expressed by neural precursors transduce a growth-regulatory signal during neurogenesis via pathways involving pertussis toxin-sensitive G-proteins, Ca2+ signalling, protein kinase C activation, MAPK phosphorylation and DNA synthesis.

  16. Anti-ageing glycoprotein promotes long-term survival of transplanted neurosensory precursor cells.

    PubMed

    Yanai, Anat; Viringipurampeer, Ishaq A; Bashar, Emran; Gregory-Evans, Kevin

    2016-05-27

    Cell therapy, to replace lost tissue, is a promising approach for the treatment of various neurodegenerative diseases. Many studies suggest, however, that the percentage of transplanted cells that survive and undergo functional integration remains low as a result of immune rejection, suboptimal precursor cell type, trauma during cell transplantation, toxic compounds released by dying tissues or nutritional deficiencies. We recently developed an ex vivo system to facilitate identification of factors contributing to the death of transplanted neuronal (photoreceptor) cells and compounds that block these toxic effects. In this system, photoreceptor precursor cells (PPCs) are sandwiched between a neurosensory retinal explant and retinal pigment epithelium derived from human embryonic stem cells. Explant medium was collected to identify toxic components and PPC survival was assessed by flow cytometry. We also assessed the potential for AAGP™, a cryopreservative molecule, to improve PPC survival. We identified elevated prostaglandin E2 (PGE2) in the explant medium and demonstrated that AAGP™ reduced PGE2 levels by 2.6-fold. A pro-inflammatory stress assay suggested that this may result from AAGP™ inhibition of cyclo-oxygenase-2 (COX-2) expression. We confirmed that PGE2 reduced the viability of cultured PPCs by 44% and found that the survival rate of PPCs pretreated with AAGP™ was 2.8-fold higher than in untreated PPCs. These data suggest that PGE2 release from necrotic tissue may be one factor that reduces the survival of transplanted precursor cells and that the pro-survival molecule AAGP™ may improve long-term transplanted cell viability. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  17. Acidity constant determination of novel drug precursor benzothiazolon derivatives including acyl and piperazine moieties

    NASA Astrophysics Data System (ADS)

    Sıdır, İsa; Gülseven Sıdır, Yadigar; Berber, Halil

    2013-07-01

    In this study, protonation and deprotonation behaviors of eight new drug precursor benzothiazolon derivatives in all of acidic and basic scale (super acidic, pH, super basic regions) are analyzed by using UV-visible spectrophotometric technique. Acidity constants (pKa), elucidation of the structure and protonation mechanisms of the studied molecules are obtained. Substituent effect on acidity constant values is discussed. These molecules are protonated from oxygen atom of acetamide group in the keto form. The protonation is found to be considerably contributed by the keto form.

  18. 9-Nitroanthracene derivative as a precursor of anthraquinone for photodynamic therapy.

    PubMed

    Fukuhara, Kiyoshi; Oikawa, Shinji; Hakoda, Nana; Sakai, Yasunori; Hiraku, Yusuke; Shoda, Takuji; Saito, Shinichi; Miyata, Naoki; Kawanishi, Shosuke; Okuda, Haruhiro

    2007-06-01

    Anthraquinones are typical photosensitizers used in photodynamic therapy (PDT). However, systemic toxicity is a major problem for anthraquinones due to their ability not only to bind DNA but also to cause oxidative stress even without photoirradiation. To avoid such disadvantages in cancer therapy, we designed and synthesized a novel 9-nitroanthracene derivative (1) as a precursor of anthraquinone. Under photoirradiation, 1 is converted into anthraquinone via generation of nitric oxide as confirmed by ESR. Strong DNA cleavage specifically at guanine under photoirradiation was also observed, characteristic of DNA-cleaving reactions by photoirradiated anthraquinones. We propose development of 1 as an alternative approach toward PDT that reduces the systemic toxicity of anthraquinone.

  19. 3-Sulfanylhexanol precursor biogenesis in grapevine cells: the stimulating effect of Botrytis cinerea.

    PubMed

    Thibon, Cécile; Cluzet, Stéphanie; Mérillon, Jean Michel; Darriet, Philippe; Dubourdieu, Denis

    2011-02-23

    Volatile thiols, compounds that contribute strongly to the varietal aroma, are present in much higher concentrations in sweet wines than in dry wines. This positive effect, due to the presence of Botrytis cinerea on the berries, in fact results from a strong enrichment of cysteine S-conjugate precursors in botrytized berries. In the present study, a convenient model was investigated to reproduce and therefore study this phenomenon. A Vitis vinifera cell culture was used as a simple model, and we focused on S-3-(hexan-1-ol)-l-cysteine (P-3SH), the cysteinylated precursor of 3-sulfanylhexanol. We demonstrated that grapevine cells were able to produce P-3SH and that the presence of B. cinerea considerably increased the precursor level (up to 1000-fold). This positive result was determined to be due to metabolites secreted by the fungus. These molecules were temperature sensitive, unstable over time, and their production was activated in the presence of grapevine cells. Moreover, part of the pathway leading to P-3SH was deciphered: it was directly derived from the cleavage of S-3-(hexan-1-ol)-l-glutathione, which itself was generated after a conjugation of glutathione on (E)-2-hexenal.

  20. Myelinogenic Plasticity of Oligodendrocyte Precursor Cells following Spinal Cord Contusion Injury.

    PubMed

    Assinck, Peggy; Duncan, Greg J; Plemel, Jason R; Lee, Michael J; Stratton, Jo A; Manesh, Sohrab B; Liu, Jie; Ramer, Leanne M; Kang, Shin H; Bergles, Dwight E; Biernaskie, Jeff; Tetzlaff, Wolfram

    2017-09-06

    Spontaneous remyelination occurs after spinal cord injury (SCI), but the extent of myelin repair and identity of the cells responsible remain incompletely understood and contentious. We assessed the cellular origin of new myelin by fate mapping platelet-derived growth factor receptor α (PDGFRα), Olig2+, and P0+ cells following contusion SCI in mice. Oligodendrocyte precursor cells (OPCs; PDGFRα+) produced oligodendrocytes responsible for de novo ensheathment of ∼30% of myelinated spinal axons at injury epicenter 3 months after SCI, demonstrating that these resident cells are a major contributor to oligodendrocyte regeneration. OPCs also produced the majority of myelinating Schwann cells in the injured spinal cord; invasion of peripheral myelinating (P0+) Schwann cells made only a limited contribution. These findings reveal that PDGFRα+ cells perform diverse roles in CNS repair, as multipotential progenitors that generate both classes of myelinating cells. This endogenous repair might be exploited as a therapeutic target for CNS trauma and disease.SIGNIFICANCE STATEMENT Spinal cord injury (SCI) leads to profound functional deficits, though substantial numbers of axons often survive. One possible explanation for these deficits is loss of myelin, creating conduction block at the site of injury. SCI leads to oligodendrocyte death and demyelination, and clinical trials have tested glial transplants to promote myelin repair. However, the degree and duration of myelin loss, and the extent and mechanisms of endogenous repair, have been contentious issues. Here, we use genetic fate mapping to demonstrate that spontaneous myelin repair by endogenous oligodendrocyte precursors is much more robust than previously recognized. These findings are relevant to many types of CNS pathology, raising the possibility that CNS precursors could be manipulated to repair myelin in lieu of glial transplantation. Copyright © 2017 the authors 0270-6474/17/378635-20$15.00/0.

  1. Derivation and spontaneous differentiation of human embryonic stem cells*

    PubMed Central

    Amit, Michal; Itskovitz-Eldor, Joseph

    2002-01-01

    Abstract Embryonic stem (ES) cells are unique cells derived from the inner cell mass of the mammalian blastocyst. These cells are immortal and pluripotent, retain their developmental potential after prolonged culture, and can be continuously cultured in an undifferentiated state. Many in vitro differentiation systems have been developed for mouse ES cells, including reproducible methods for mouse ES cell differentiation into haematopoietic and neural precursors, cardiomyocytes, insulin-secreting cells, endothelial cells and various other cell types. The derivation of new human ES cell lines provides the opportunity to develop unique models for developmental research and for cell therapies. In this review we consider the derivation and spontaneous differentiation of human ES cells. PMID:12033726

  2. GBM secretome induces transient transformation of human neural precursor cells.

    PubMed

    Venugopal, Chitra; Wang, X Simon; Manoranjan, Branavan; McFarlane, Nicole; Nolte, Sara; Li, Meredith; Murty, Naresh; Siu, K W Michael; Singh, Sheila K

    2012-09-01

    Glioblastoma (GBM) is the most aggressive primary brain tumor in humans, with a uniformly poor prognosis. The tumor microenvironment is composed of both supportive cellular substrates and exogenous factors. We hypothesize that exogenous factors secreted by brain tumor initiating cells (BTICs) could predispose normal neural precursor cells (NPCs) to transformation. When NPCs are grown in GBM-conditioned media, and designated as "tumor-conditioned NPCs" (tcNPCs), they become highly proliferative and exhibit increased stem cell self-renewal, or the unique ability of stem cells to asymmetrically generate another stem cell and a daughter cell. tcNPCs also show an increased transcript level of stem cell markers such as CD133 and ALDH and growth factor receptors such as VEGFR1, VEGFR2, EGFR and PDGFRα. Media analysis by ELISA of GBM-conditioned media reveals an elevated secretion of growth factors such as EGF, VEGF and PDGF-AA when compared to normal neural stem cell-conditioned media. We also demonstrate that tcNPCs require prolonged or continuous exposure to the GBM secretome in vitro to retain GBM BTIC characteristics. Our in vivo studies reveal that tcNPCs are unable to form tumors, confirming that irreversible transformation events may require sustained or prolonged presence of the GBM secretome. Analysis of GBM-conditioned media by mass spectrometry reveals the presence of secreted proteins Chitinase-3-like 1 (CHI3L1) and H2A histone family member H2AX. Collectively, our data suggest that GBM-secreted factors are capable of transiently altering normal NPCs, although for retention of the transformed phenotype, sustained or prolonged secretome exposure or additional transformation events are likely necessary.

  3. Early T-cell precursor acute lymphoblastic leukaemia.

    PubMed

    Haydu, J Erika; Ferrando, Adolfo A

    2013-07-01

    Early T-cell precursor (ETP) leukaemias have been recently recognized as a form of T-cell acute lymphoblastic leukaemia (T-ALL) with a poor prognosis. The purpose of this review is to outline the most recent advances in the biology, genetics and prognostic significance of this aggressive disease. Detailed immunophenotypic analyses have defined ETP T-ALLs as a distinct group of T-ALL with a poor prognosis. Transcriptionally, ETP T-ALLs and early immature T-ALLs, a broader group of tumours characterized by very early arrest in T-cell differentiation, are most related to haematopoietic stem cells and myeloid progenitors. Consistently, these leukaemias show lower frequencies of prototypical T-ALL lesions such as CDKN2A/B deletions and activating mutations in NOTCH1 and show a higher prevalence of mutations typically associated with the pathogenesis of acute myeloid leukaemias (AMLs). ETP and early immature T-ALLs are characterized by a very early differentiation arrest and show unique genetic and transcriptional features that overlap both with T-ALL and with AML. Given the unique biology and poor prognosis associated with the ETP T-ALL group, there is an urgent need of new tailored therapeutic strategies for the treatment of this disease.

  4. Precursors of executive function in infants with sickle cell anemia.

    PubMed

    Hogan, Alexandra M; Telfer, Paul T; Kirkham, Fenella J; de Haan, Michelle

    2013-10-01

    Executive dysfunction occurs in sickle cell anemia, but there are few early data. Infants with sickle cell anemia (n = 14) and controls (n = 14) performed the "A-not-B" and Object Retrieval search tasks, measuring precursors of executive function at 9 and 12 months. Significant group differences were not found. However, for the A-not-B task, 7 of 11 sickle cell anemia infants scored in the lower 2 performance categories at 9 months, but only 1 at 12 months (P = .024); controls obtained scores at 12 months that were statistically comparable to the scores they had already obtained at 9 months. On the Object Retrieval task, 9- and 12-month controls showed comparable scores, whereas infants with sickle cell anemia continued to improve (P = .027); at 9 months, those with lower hemoglobin oxygen saturation passed fewer trials (R s = 0.670, P = .024) and took longer to obtain the toy (R s = -0.664, P = .013). Subtle delays in acquiring developmental skills may underlie abnormal executive function in childhood.

  5. Generation of Valpha14 NKT cells in vitro from hematopoietic precursors residing in bone marrow and peripheral blood.

    PubMed

    Shimamura, Michio; Kobayashi, Kumi; Watanabe, Hiroko; Huang, Yi-Ying; Okamoto, Naoki; Kanie, Osamu; Goji, Hiroshi; Kobayashi, Masumi

    2004-03-01

    We previously reported the generation of Valpha14 invariant TCR+ (Valpha14i) NK1.1+ natural killer T (NKT) cells in the cytokine-activated suspension culture of murine fetal liver cells. In this study, we attempted to apply this finding to the induction of Valpha14i NKT cell differentiation in the culture of hematopoietic precursors residing in bone marrow or peripheral blood. Preferential generation of NKT cells was found in the culture of Thy-1(+)-depleted bone marrow cells in the presence of culture supernatant from Con A-stimulated spleen T cells and a combination of recombinant IL-3, IL-4, IL-7 and GM-CSF. NKT cell development from peripheral blood hematopoietic precursors was induced when they were cultured on stromal cell monolayers prepared from Thy-1(+)-depleted bone marrow or fetal liver cells, suggesting that certain environments derived from hematopoietic organs are required for the induction of NKT cells from precursors in vitro. A significant fraction of NKT cells generated in the culture were positive for staining with CD1-alpha-galactosylceramide tetramer, indicating that Valpha14i NKT cells were the major subset among the NKT cells. The present methods for obtaining NKT cells in the culture of bone marrow or peripheral blood cells are applicable to the treatment of patients suffering from diseases with numerical and functional disorders of NKT cells.

  6. Integrin-mediated interactions between human bone marrow stromal precursor cells and the extracellular matrix.

    PubMed

    Gronthos, S; Simmons, P J; Graves, S E; Robey, P G

    2001-02-01

    To date, the precise interactions between bone marrow stromal cells and the extracellular matrix that govern stromal cell development remain unclear. The integrin super-family of cell-surface adhesion molecules represents a major pathway used by virtually all cell types to interact with different extracellular matrix components. In this study, purified populations of stromal precursor cells were isolated from the STRO-1-positive fraction of normal human marrow, by fluoresence-activated cell sorting, and then assayed for their ability to initiate clonogenic growth in the presence of various integrin ligands. Bone marrow-derived stromal progenitors displayed differential growth to fibronectin, vitronectin, and laminin, over collagen types I and III, but showed a similar affinity for collagen type IV. The integrin heterodimers alpha1beta1, alpha2beta1, alpha5beta1, alpha6beta1, alpha(v)beta3, and alpha(v)beta5 were found to coexpress with the STRO-1 antigen on the cell surface of CFU-F, using dual-color analysis. Furthermore, only a proportion of stromal precursors expressed the integrin alpha4beta1, while no measurable levels of the integrin alpha3beta1 could be detected. Subsequent adhesion studies using functional blocking antibodies to different integrin alpha/beta heterodimers showed that stromal cell growth on collagen, laminin, and fibronectin was mediated by multiple beta1 integrins. In contrast, cloning efficiency in the presence of vitronectin was mediated in part by alpha(v)beta3. When human marrow stromal cells were cultured under osteoinductive conditions, their ability to form a mineralized matrix in vitro was significantly diminished in the presence of a functional blocking monoclonal antibody to the beta1 integrin subunit. The results of this study indicate that beta1 integrins appear to be the predominant adhesion receptor subfamily utilized by stromal precursor cells to adhere and proliferate utilizing matrix glycoproteins commonly found in the bone

  7. Skin-derived precursors possess the ability of differentiation into the epidermal progeny and accelerate burn wound healing.

    PubMed

    Bayati, Vahid; Abbaspour, Mohammad Reza; Neisi, Niloofar; Hashemitabar, Mahmoud

    2017-02-01

    Skin-derived precursors (SKPs) are remnants of the embryonic neural crest stem cells that reside in the dermis until adulthood. Although they possess a wide range of differentiation potentials, their differentiation into keratinocyte-like cells and their roles in skin wound healing are obscure. The present study aimed to investigate the differentiation of SKPs into keratinocyte-like cells and evaluate their role in healing of third degree burn wounds. To this aim, SKPs were differentiated into keratinocyte-like cells on tissue culture plate and collagen-chitosan scaffold prepared by freeze-drying. Their differentiation capability was detected by real-time RT-PCR and immunofluorescence. Thereafter, they were cultured on scaffold and implanted in a rat model of burn wound. Histopathological and immunohistochemical analyses were employed to examine the reconstituted skin. The research findings revealed that SKPs were able to differentiate along the epidermal lineage and this ability can be enhanced on a suitable scaffold. Additionally, the results indicated that SKPs apparently promoted wound healing process and accelerate its transition from proliferating stage to maturational phase, especially if they were differentiated into keratinocyte-like cells. Regarding the results, it is concluded that SKPs are able to differentiate into keratinocyte-like cells, particularly when they are cultured on collagen-chitosan scaffold. Moreover, they can regenerate epidermal and dermal layers including thick collagen bundles, possibly through differentiation into keratinocyte-like cells.

  8. A monoclonal antibody that recognizes B cells and B cell precursors in mice

    PubMed Central

    1981-01-01

    The monoclonal antibody, RA3-2C2, appears to be specific for cells within the B cell lineage. This antibody does not recognize thymocytes, peripheral T cells, or nonlymphoid hematopoietic cells in the spleen or bone marrow. Nor does it recognize the pluripotent hematopoietic stem cells, the spleen colony-forming unit, All sIg+ B cells and most plasma cells are RA3-2C2+. In addition, approximately 20% of nucleated bone marrow cells are RA3-2C2+ but sIg-. This population contains B cell precursors that can give rise to sIg+ cells within 2 d in vitro. PMID:6787164

  9. A monoclonal antibody that recognizes B cells and B cell precursors in mice

    SciTech Connect

    Coffman, R.L.; Weissman, I.L.

    1981-02-01

    The monoclonal antibody, RA3-2C2, appears to be specific for cells within the B cell lineage. This antibody does not recognize thymocytes, peripheral T cells, or nonlymphoid hematopoietic cells in the spleen or bone marrow. Nor does it recognize the pluripotent hematopoietic stem cells, the spleen colony-forming unit, All sIg+ B cells and most plasma cells are RA3-2C2+. In addition, approximately 20% of nucleated bone marrow cells are RA3-2C2+ but sIg-. This population contains B cell precursors that can give rise to sIg+ cells within 2 d in vitro.

  10. Secretory clusterin inhibits osteoclastogenesis by attenuating M-CSF-dependent osteoclast precursor cell proliferation

    SciTech Connect

    Choi, Bongkun; Kang, Soon-Suk; Kang, Sang-Wook; Min, Bon-Hong; Lee, Eun-Jin; Song, Da-Hyun; Kim, Sang-Min; Song, Youngsup; Yoon, Seung-Yong; Chang, Eun-Ju

    2014-07-18

    Highlights: • We describe the expression and secretion of clusterin in osteoclasts. • Endogenous clusterin deficiency does not affect osteoclast formation. • Exogenous treatment with secretory clusterin decreases osteoclast differentiation. • Secretory clusterin attenuates osteoclast precursor cell proliferation by inhibiting M-CSF-mediated ERK activation. - Abstract: Secretory clusterin (sCLU)/apolipoprotein J is a multifunctional glycoprotein that is ubiquitously expressed in various tissues. Reduced sCLU in the joints of patients with bone erosive disease is associated with disease activity; however, its exact role has yet to be elucidated. Here, we report that CLU is expressed and secreted during osteoclastogenesis in mouse bone marrow-derived macrophages (BMMs) that are treated with receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). CLU-deficient BMMs obtained from CLU{sup −/−} mice exhibited no significant alterations in OC differentiation in comparison with BMMs obtained from wild-type mice. In contrast, exogenous sCLU treatment significantly inhibited OC formation in both BMMs and OC precursor cultures. The inhibitory effect of sCLU was more prominent in BMMs than OC precursor cultures. Interestingly, treating BMMs with sCLU decreased the proliferative effects elicited by M-CSF and suppressed M-CSF-induced ERK activation of OC precursor cells without causing apoptotic cell death. This study provides the first evidence that sCLU reduces OC formation by inhibiting the actions of M-CSF, thereby suggesting its protective role in bone erosion.

  11. Lysophosphatidic Acid Receptor Is a Functional Marker of Adult Hippocampal Precursor Cells.

    PubMed

    Walker, Tara L; Overall, Rupert W; Vogler, Steffen; Sykes, Alex M; Ruhwald, Susann; Lasse, Daniela; Ichwan, Muhammad; Fabel, Klaus; Kempermann, Gerd

    2016-04-12

    Here, we show that the lysophosphatidic acid receptor 1 (LPA1) is expressed by a defined population of type 1 stem cells and type 2a precursor cells in the adult mouse dentate gyrus. LPA1, in contrast to Nestin, also marks the quiescent stem cell population. Combining LPA1-GFP with EGFR and prominin-1 expression, we have enabled the prospective separation of both proliferative and non-proliferative precursor cell populations. Transcriptional profiling of the isolated proliferative precursor cells suggested immune mechanisms and cytokine signaling as molecular regulators of adult hippocampal precursor cell proliferation. In addition to LPA1 being a marker of this important stem cell population, we also show that the corresponding ligand LPA is directly involved in the regulation of adult hippocampal precursor cell proliferation and neurogenesis, an effect that can be attributed to LPA signaling via the AKT and MAPK pathways. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  12. Attenuation of proliferation in oligodendrocyte precursor cells by activated microglia.

    PubMed

    Taylor, Deanna L; Pirianov, Grisha; Holland, Samantha; McGinnity, Colm J; Norman, Adele L; Reali, Camilla; Diemel, Lara T; Gveric, Djordje; Yeung, Davy; Mehmet, Huseyin

    2010-06-01

    Activated microglia can influence the survival of neural cells through the release of cytotoxic factors. Here, we investigated the interaction between Toll-like receptor 4 (TLR4)-activated microglia and oligodendrocytes or their precursor cells (OPC). Primary rat or N9 microglial cells were activated by exposure to TLR4-specifc lipopolysaccharide (LPS), resulting in mitogen-activated protein kinase activation, increased CD68 and inducible nitric oxide synthase expression, and release of the proinflammatory cytokines tumor necrosis factor (TNF) and interleukin-6 (IL-6). Microglial conditioned medium (MGCM) from LPS-activated microglia attenuated primary OPC proliferation without inducing cell death. The microglial-induced inhibition of OPC proliferation was reversed by stimulating group III metabotropic glutamate receptors in microglia with the agonist L-AP4. In contrast to OPC, LPS-activated MGCM enhanced the survival of mature oligodendrocytes. Further investigation suggested that TNF and IL-6 released from TLR4-activated microglia might contribute to the effect of MGCM on OPC proliferation, insofar as TNF depletion of LPS-activated MGCM reduced the inhibition of OPC proliferation, and direct addition of TNF or IL-6 attenuated or increased proliferation, respectively. OPC themselves were also found to express proteins involved in TLR4 signalling, including TLR4, MyD88, and MAL. Although LPS stimulation of OPC did not induce proinflammatory cytokine release or affect their survival, it did trigger JNK phosphorylation, suggesting that TLR4 signalling in these cells is active. These findings suggest that OPC survival may be influenced not only by factors released from endotoxin-activated microglia but also through a direct response to endotoxins. This may have consequences for myelination under conditions in which microglial activation and cerebral infection are both implicated. , Inc.

  13. Circulating Osteogenic Precursor Cells in Type 2 Diabetes Mellitus

    PubMed Central

    Manavalan, J. S.; Cremers, S.; Dempster, D. W.; Zhou, H.; Dworakowski, E.; Kode, A.; Kousteni, S.

    2012-01-01

    Context: Type 2 diabetes mellitus (T2D) is associated with an increased risk of fractures and low bone formation. However, the mechanism for the low bone formation is not well understood. Recently, circulating osteogenic precursor (COP) cells, which contribute to bone formation, have been characterized in the peripheral circulation. Objective: Our objective was to characterize the number and maturity of COP cells in T2D. Patients, Design, and Setting: Eighteen postmenopausal women with T2D and 27 controls participated in this cross-sectional study at a clinical research center. Main Outcome Measures: COP cells were characterized using flow cytometry and antibodies against osteocalcin (OCN) and early stem cell markers. Histomorphometric (n = 9) and molecular (n=14) indices of bone turnover and oxidative stress were also measured. Results: The percentage of OCN+ cells in peripheral blood mononuclear cells was lower in T2D (0.8 ± 0.2 vs. 1.6 ± 0.4%; P < 0.0001), whereas the percentage of OCN+ cells coexpressing the early marker CD146 was increased (OCN+/CD146+: 33.3 ± 7 vs. 12.0 ± 4%; P < 0.0001). Reduced histomorphometric indices of bone formation were observed in T2D subjects, including mineralizing surface (2.65 ± 1.9 vs. 7.58 ± 2.4%, P = 0.02), bone formation rate (0.01 ± 0.1 vs. 0.05 ±0.2 μm3/um2 · d, P = 0.02), and osteoblast surface (1.23 ±0.9 vs. 4.60 ± 2.5%, P = 0.03). T2D subjects also had reduced molecular expression of the osteoblast regulator gene Runx2 but increased expression of the oxidative stress markers p66Shc and SOD2. Conclusions: Circulating OCN+ cells were decreased in T2D, whereas OCN+/CD146+ cells were increased. Histomorphometric indices of bone formation were decreased in T2D, as was molecular expression of osteoblastic activity. Stimulation of bone formation may have beneficial therapeutic skeletal consequences in T2D. PMID:22740707

  14. Ex Situ Formation of Metal Selenide Quantum Dots Using Bacterially Derived Selenide Precursors

    SciTech Connect

    Fellowes, Jonathan W.; Pattrick, Richard; Lloyd, Jon; Charnock, John M.; Coker, Victoria S.; Mosselmans, JFW; Weng, Tsu-Chien; Pearce, Carolyn I.

    2013-04-12

    Luminescent quantum dots were synthesized using bacterially derived selenide (SeII-) as the precursor. Biogenic SeII- was produced by the reduction of Se-IV by Veillonella atypica and compared directly against borohydride-reduced Se-IV for the production of glutathione-stabilized CdSe and beta-mercaptoethanol-stabilized ZnSe nanoparticles by aqueous synthesis. Biological SeII- formed smaller, narrower size distributed QDs under the same conditions. The growth kinetics of biologically sourced CdSe phases were slower. The proteins isolated from filter sterilized biogenic SeII- included a methylmalonyl-CoA decarboxylase previously characterized in the closely related Veillonella parvula. XAS analysis of the glutathione-capped CdSe at the S K-edge suggested that sulfur from the glutathione was structurally incorporated within the CdSe. A novel synchrotron based XAS technique was also developed to follow the nucleation of biological and inorganic selenide phases, and showed that biogenic SeII- is more stable and more resistant to beam-induced oxidative damage than its inorganic counterpart. The bacterial production of quantum dot precursors offers an alternative, 'green' synthesis technique that negates the requirement of expensive, toxic chemicals and suggests a possible link to the exploitation of selenium contaminated waste streams.

  15. Ex situ formation of metal selenide quantum dots using bacterially derived selenide precursors.

    PubMed

    Fellowes, J W; Pattrick, R A D; Lloyd, J R; Charnock, J M; Coker, V S; Mosselmans, J F W; Weng, T-C; Pearce, C I

    2013-04-12

    Luminescent quantum dots were synthesized using bacterially derived selenide (Se(II-)) as the precursor. Biogenic Se(II-) was produced by the reduction of Se(IV) by Veillonella atypica and compared directly against borohydride-reduced Se(IV) for the production of glutathione-stabilized CdSe and β-mercaptoethanol-stabilized ZnSe nanoparticles by aqueous synthesis. Biological Se(II-) formed smaller, narrower size distributed QDs under the same conditions. The growth kinetics of biologically sourced CdSe phases were slower. The proteins isolated from filter sterilized biogenic Se(II-) included a methylmalonyl-CoA decarboxylase previously characterized in the closely related Veillonella parvula. XAS analysis of the glutathione-capped CdSe at the S K-edge suggested that sulfur from the glutathione was structurally incorporated within the CdSe. A novel synchrotron based XAS technique was also developed to follow the nucleation of biological and inorganic selenide phases, and showed that biogenic Se(II-) is more stable and more resistant to beam-induced oxidative damage than its inorganic counterpart. The bacterial production of quantum dot precursors offers an alternative, 'green' synthesis technique that negates the requirement of expensive, toxic chemicals and suggests a possible link to the exploitation of selenium contaminated waste streams.

  16. Gradient isolation of glial cells: evidence that flat epithelial cells are astroglial cell precursors.

    PubMed

    Meller, K

    1987-07-01

    Discontinuous gradients of metrizamide were used to separate the cell components of monolayers of primary cultures of embryonic rat brains. These primary cell cultures were of two types: long-term cultures (more than a year) of embryonic rat brain, which contained several glial cell types, and monolayers of cell cultures (several weeks old), which contained a complex population of cells, including neuronal elements. The gradient separation produces fractions of pure flat epithelial cells that are able to survive and proliferate. After a few days, all flat epithelial cells become confluent and show a positive reaction to glial fibrillary acidic protein (GFAP); this indicates that these cells astroglial precursor cells. Following their maintenance in vitro for several months, all cultures give rise to a pure population of astrocytes identified not only by their characteristic morphology, but also by their content of GFAP. It is proposed that the differentiation controls are dependent on cell interactions that are influenced by the composition of the cell population and/or the molecular growth and differentiation factors released by these cells into the medium.

  17. Archenteron precursor cells can organize secondary axial structures in the sea urchin embryo.

    PubMed

    Benink, H; Wray, G; Hardin, J

    1997-09-01

    Local cell-cell signals play a crucial role in establishing major tissue territories in early embryos. The sea urchin embryo is a useful model system for studying these interactions in deuterostomes. Previous studies showed that ectopically implanted micromeres from the 16-cell embryo can induce ectopic guts and additional skeletal elements in sea urchin embryos. Using a chimeric embryo approach, we show that implanted archenteron precursors differentiate autonomously to produce a correctly proportioned and patterned gut. In addition, the ectopically implanted presumptive archenteron tissue induces ectopic skeletal patterning sites within the ectoderm. The ectopic skeletal elements are bilaterally symmetric, and flank the ectopic archenteron, in some cases resulting in mirror-image, symmetric skeletal elements. Since the induced patterned ectoderm and supernumerary skeletal elements are derived from the host, the ectopic presumptive archenteron tissue can act to 'organize' ectopic axial structures in the sea urchin embryo.

  18. Adult neurogenesis in the crayfish brain: proliferation, migration, and possible origin of precursor cells.

    PubMed

    Zhang, Yi; Allodi, Silvana; Sandeman, David C; Beltz, Barbara S

    2009-06-01

    The birth of new neurons and their incorporation into functional circuits in the adult brain is a characteristic of many vertebrate and invertebrate organisms, including decapod crustaceans. Precursor cells maintaining life-long proliferation in the brains of crayfish (Procambarus clarkii, Cherax destructor) and clawed lobsters (Homarus americanus) reside within a specialized niche on the ventral surface of the brain; their daughters migrate to two proliferation zones along a stream formed by processes of the niche precursors. Here they divide again, finally producing interneurons in the olfactory pathway. The present studies in P. clarkii explore (1) differential proliferative activity among the niche precursor cells with growth and aging, (2) morphological characteristics of cells in the niche and migratory streams, and (3) aspects of the cell cycle in this lineage. Morphologically symmetrical divisions of neuronal precursor cells were observed in the niche near where the migratory streams emerge, as well as in the streams and proliferation zones. The nuclei of migrating cells elongate and undergo shape changes consistent with nucleokinetic movement. LIS1, a highly conserved dynein-binding protein, is expressed in cells in the migratory stream and neurogenic niche, implicating this protein in the translocation of crustacean brain neuronal precursor cells. Symmetrical divisions of the niche precursors and migration of both daughters raised the question of how the niche precursor pool is replenished. We present here preliminary evidence for an association between vascular cells and the niche precursors, which may relate to the life-long growth and maintenance of the crustacean neurogenic niche.

  19. Cell cycle regulation during proliferation and differentiation of mammalian muscle precursor cells.

    PubMed

    Ciemerych, Maria A; Archacka, Karolina; Grabowska, Iwona; Przewoźniak, Marta

    2011-01-01

    Proliferation and differentiation of muscle precursor cells are intensively studied not only in the developing mouse embryo but also using models of skeletal muscle regeneration or analyzing in vitro cultured cells. These analyses allowed to show the universality of the cell cycle regulation and also uncovered tissue-specific interplay between major cell cycle regulators and factors crucial for the myogenic differentiation. Examination of the events accompanying proliferation and differentiation leading to the formation of functional skeletal muscle fibers allows understanding the molecular basis not only of myogenesis but also of skeletal muscle regeneration. This chapter presents the basis of the cell cycle regulation in proliferating and differentiating muscle precursor cells during development and after muscle injury. It focuses at major cell cycle regulators, myogenic factors, and extracellular environment impacting on the skeletal muscle.

  20. A multipurpose fusion tag derived from an unstructured and hyperacidic region of the amyloid precursor protein

    PubMed Central

    Sangawa, Takeshi; Tabata, Sanae; Suzuki, Kei; Saheki, Yasushi; Tanaka, Keiji; Takagi, Junichi

    2013-01-01

    Expression and purification of aggregation-prone and disulfide-containing proteins in Escherichia coli remains as a major hurdle for structural and functional analyses of high-value target proteins. Here, we present a novel gene-fusion strategy that greatly simplifies purification and refolding procedure at very low cost using a unique hyperacidic module derived from the human amyloid precursor protein. Fusion with this polypeptide (dubbed FATT for Flag-Acidic-Target Tag) results in near-complete soluble expression of variety of extracellular proteins, which can be directly refolded in the crude bacterial lysate and purified in one-step by anion exchange chromatography. Application of this system enabled preparation of functionally active extracellular enzymes and antibody fragments without the need for condition optimization. PMID:23526492

  1. On electric conduction of amorphous silicon carbonitride derived from a polymeric precursor

    NASA Astrophysics Data System (ADS)

    Chen, Yaohan; Yang, Fuqian; An, Linan

    2013-06-01

    A long-existing problem that the conductivity of amorphous silicon carbonitrides derived from polymeric precursor increases significantly with pyrolysis temperature is investigated. We show that the conductivity exhibited an Arrhenius dependence on pyrolysis temperature, with the activation energy of ˜3.41 eV. Structural analysis using Raman spectroscopy reveals that the free carbon within the material undergoes a sp3-to-sp2 transition as pyrolysis temperature increases, with the activation energy of ˜3.6 eV. We conclude that the pyrolysis-temperature induced increase in the conductivity is mainly due to the increase in the conductivity of the free carbon. A simple model is proposed to correlate the two.

  2. Role of precursor crystal structure on electrochemical performance of carbide-derived carbon electrodes

    NASA Astrophysics Data System (ADS)

    Palazzo, Benjamin; Norris, Zach; Taylor, Greg; Yu, Lei; Lofland, Samuel; Hettinger, Jeffrey

    2015-03-01

    Binary carbides with hexagonal and cubic crystal structures have been synthesized by reactive magnetron sputtering of vanadium and other transition metals in acetylene or methane gas mixed with argon. The binary carbides are converted to carbide-derived carbon (CDC) films using chlorine gas in a post-deposition process in an external vacuum reaction furnace. Residual chlorine has been removed using an annealing step in a hydrogen atmosphere. The CDC materials have been characterized by x-ray diffraction, x-ray fluorescence, and scanning electron microscopy. The performance of the CDC materials in electrochemical device applications has been measured with the hexagonal phase precursor demonstrating a significantly higher specific capacitance in comparison to that of the cubic phase. We report these results and pore-size distributions of these and similar materials.

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

    PubMed

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

    2016-12-05

    In the last years, mesenchymal stromal cells (MSCs) from oral tissues have received considerable interest in regenerative medicine since they can be obtained with minimal invasive procedure and exhibit immunomodulatory properties. This study was aimed to investigate whether in vitro pre-treatment of MSCs obtained from human gingiva (hGMSCs) with Cannabidiol (CBD), a cannabinoid component produced by the plant Cannabis sativa, may promote human gingiva derived MSCs to differentiate toward neuronal precursor cells. Specifically, we have treated the hGMSCs with CBD (5 µM) for 24 h in order to evaluate the expression of genes involved in cannabidiol signaling, cell proliferation, self-renewal and multipotency, and neural progenitor cells differentiation. Next generation sequencing (NGS) demonstrated that CBD activates genes associated with G protein coupled receptor signaling in hGMSCs. Genes involved in DNA replication, cell cycle, proliferation, and apoptosis were regulated. Moreover, genes associated with the biological process of neuronal progenitor cells (NCPs) proliferation, neuron differentiation, neurogenesis, and nervous system development were significantly modulated. From our results, we hypothesize that human gingiva-derived MSCs conditioned with CBD could represent a valid method for improving the hGMSCs phenotype and thus might be a potential therapeutic tool in the treatment of neurodegenerative diseases. J. Cell. Biochem. 9999: 1-16, 2016. © 2016 Wiley Periodicals, Inc.

  4. CD44-positive cells are candidates for astrocyte precursor cells in developing mouse cerebellum.

    PubMed

    Cai, Na; Kurachi, Masashi; Shibasaki, Koji; Okano-Uchida, Takayuki; Ishizaki, Yasuki

    2012-03-01

    Neural stem cells are generally considered to be committed to becoming precursor cells before terminally differentiating into either neurons or glial cells during neural development. Neuronal and oligodendrocyte precursor cells have been identified in several areas in the murine central nervous system. The presence of astrocyte precursor cells (APCs) is not so well understood. The present study provides several lines of evidence that CD44-positive cells are APCs in the early postnatal mouse cerebellum. In developing mouse cerebellum, CD44-positive cells, mostly located in the white matter, were positive for the markers of the astrocyte lineage, but negative for the markers of mature astrocytes. CD44-positive cells were purified from postnatal cerebellum by fluorescence-activated cell sorting and characterized in vitro. In the absence of any signaling molecule, many cells died by apoptosis. The surviving cells gradually expressed glial fibrillary acidic protein, a marker for mature astrocytes, indicating that differentiation into mature astrocytes is the default program for these cells. The cells produced no neurospheres nor neurons nor oligodendrocytes under any condition examined, indicating these cells are not neural stem cells. Leukemia inhibitory factor greatly promoted astrocytic differentiation of CD44-positive cells, whereas bone morphogenetic protein 4 (BMP4) did not. Fibroblast growth factor-2 was a potent mitogen for these cells, but was insufficient for survival. BMP4 inhibited activation of caspase-3 and greatly promoted survival, suggesting a novel role for BMP4 in the control of development of astrocytes in cerebellum. We isolated and characterized only CD44 strongly positive large cells and discarded small and/or CD44 weakly positive cells in this study. Further studies are necessary to characterize these cells to help determine whether CD44 is a selective and specific marker for APCs in the developing mouse cerebellum. In conclusion, we succeeded in

  5. Bone marrow B cell precursor number after allogeneic stem cell transplantation and GVHD development.

    PubMed

    Fedoriw, Yuri; Samulski, T Danielle; Deal, Allison M; Dunphy, Cherie H; Sharf, Andrew; Shea, Thomas C; Serody, Jonathan S; Sarantopoulos, Stefanie

    2012-06-01

    Patients without chronic graft-versus-host disease (cGVHD) have robust B cell reconstitution and are able to maintain B cell homeostasis after allogeneic hematopoietic stem cell transplantation (HSCT). To determine whether B lymphopoiesis differs before cGVHD develops, we examined bone marrow (BM) biopsies for terminal deoxynucleotidyl transferase (TdT) and PAX5 immunostaining early post-HSCT at day 30 when all patients have been shown to have high B cell activating factor (BAFF) levels. We found significantly greater numbers of BM B cell precursors in patients who did not develop cGVHD compared with those who developed cGVHD (median = 44 vs 2 cells/high powered field [hpf]; respectively; P < .001). Importantly, a significant increase in precursor B cells was maintained when patients receiving high-dose steroid therapy were excluded (median = 49 vs 20 cells/hpf; P = .017). Thus, we demonstrate the association of BM B cell production capacity in human GVHD development. Increased BM precursor B cell number may serve to predict good clinical outcome after HSCT. Copyright © 2012 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

  6. Bone Marrow B cell Precursor Number after Allogeneic Stem Cell Transplantation and GVHD Development

    PubMed Central

    Fedoriw, Yuri; Samulski, T. Danielle; Deal, Allison M.; Dunphy, Cherie H.; Sharf, Andrew; Shea, Thomas C.; Serody, Jonathan S.; Sarantopoulos, Stefanie

    2013-01-01

    Patients without chronic graft-versus-host disease (cGVHD) have robust B cell reconstitution and are able to maintain B cell homeostasis after allogeneic hematopoietic stem cell transplantation (HSCT). To determine whether B lymphopoiesis differs before cGVHD develops, we examined bone marrow (BM) biopsies for terminal deoxynucleotidyl transferase (TdT) and PAX5 immunostaining early post-HSCTat day 30 when all patients have been shown to have high B cell activating factor (BAFF) levels. We found significantly greater numbers of BM B cell precursors in patients who did not develop cGVHD compared with those who developed cGVHD (median = 44 vs 2 cells/high powered field [hpf]; respectively; P < .001). Importantly, a significant increase in precursor B cells was maintained when patients receiving high-dose steroid therapy were excluded (median = 49 vs 20 cells/hpf; P =.017). Thus, we demonstrate the association of BM B cell production capacity in human GVHD development. Increased BM precursor B cell number may serve to predict good clinical outcome after HSCT. PMID:22446015

  7. [Regenerative potential of human adult precursor cells: cell therapy--an option for treating cartilage defects?].

    PubMed

    Dehne, T; Tschirschmann, M; Lauster, R; Sittinger, M

    2009-05-01

    Cell-based therapeutical approaches are already in clinical use and are attracting growing interest for the treatment of joint defects. Mesenchymal stem and precursor cells (MSC) cover a wide range of properties that are useful for the regeneration process of bone and cartilage defects. The following article is an overview of the regenerative potential of MSC and discusses how the properties of these cells can be used for the development of new strategies in regenerative medicine.

  8. Characterization of the Murine Myeloid Precursor Cell Line MuMac-E8

    PubMed Central

    Fricke, Stephan; Riemschneider, Sina; Kohlschmidt, Janine; Hilger, Nadja; Fueldner, Christiane; Knauer, Jens; Sack, Ulrich; Emmrich, Frank; Lehmann, Jörg

    2014-01-01

    Starting point for the present work was the assumption that the cell line MuMac-E8 represents a murine cell population with stem cell properties. Preliminary studies already pointed to the expression of stem-cell associated markers and a self-regenerative potential of the cells. The cell line MuMac-E8 should be examined for their differential stage within stem cell hierarchy. MuMac-E8 cells were derived from a chimeric mouse model of arthritis. It could be shown that MuMac-E8 cells express mRNA of some genes associated with pluripotent stem cells (Nanog, Nucleostemin), of genes for hematopoietic markers (EPCR, Sca-1, CD11b, CD45), for the mesenchymal marker CD105 and of genes for the neural markers Pax-6 and Ezrin. In methylcellulose and May-Grünwald-Giemsa staining, hematopoietic colonies were obtained but the hematopoietic system of lethally irradiated mice could not be rescued. Osteogenic differentiation was not detectable. Thus, it became evident that MuMac-E8 represents not a stem cell line. However, MuMac-E8 cells expressed several myeloid surface markers (i.e. CD11b, F4/80, CD14, CD64), showed phagocytosis and is capable of producing nitric oxide. Thus, this cell line seems to be arrested an advanced stage of myeloid differentiation. Adherence data measured by impedance-based real-time cell analysis together with cell morphology data suggested that MuMac-E8 represents a new macrophage precursor cell line exhibiting weak adherence. This cell line is suitable as an in-vitro model for testing of macrophage functions. Moreover, it might be also useful for differentiation or reprogramming studies. PMID:25546418

  9. Cell-type dependent modulation of Notch signaling by the amyloid precursor protein.

    PubMed

    Oh, Sun Young; Chen, Ci-Di; Abraham, Carmela R

    2010-04-01

    The amyloid precursor protein is a ubiquitously expressed transmembrane protein that has been long implicated in the pathogenesis of Alzheimer's disease but its normal biological function has remained elusive despite extensive effort. We have previously reported the identification of Notch2 as an amyloid precursor protein interacting protein in E18 rat neurons. Here, we sought to reveal the physiologic consequences of this interaction. We report a functional relationship between amyloid precursor protein and Notch1, which does not affect Delta ligand binding. First, we observed interactions between the amyloid precursor protein and Notch in mouse embryonic stem cells lacking both presenilin 1 and presenilin 2, the active proteolytic components of the gamma-secretase complex, suggesting that these two transmembrane proteins can interact in the absence of presenilin. Next, we demonstrated that the amyloid precursor protein affects Notch signaling by using Notch-dependent luciferase assays in two cell lines, the human embryonic kidney 293 and the monkey kidney, COS7. We found that the amyloid precursor protein exerts opposing effects on Notch signaling in human embryonic kidney 293 vs. COS7 cells. Finally, we show that more Notch Intracellular Domain is found in the nucleus in the presence of exogenous amyloid precursor protein or its intracellular domain, suggesting the mechanism by which the amyloid precursor protein affects Notch signaling in certain cells. Our results provide evidence of potentially important communications between the amyloid precursor protein and Notch.

  10. Expression of CD86 on human marrow CD34(+) cells identifies immunocompetent committed precursors of macrophages and dendritic cells.

    PubMed

    Ryncarz, R E; Anasetti, C

    1998-05-15

    Macrophages and dendritic cells derive from a hematopoietic stem cell and the existence of a common committed progenitor has been hypothesized. We have recently found in normal human marrow a subset of CD34(+) cells that constitutively expresses HLA-DR and low levels of CD86, a natural ligand for the T cell costimulation receptor CD28. This CD34(+) subset can elicit responses from allogeneic T cells. In this study, we show that CD34(+)/CD86(+) cells can also present tetanus toxoid antigen to memory CD4(+) T cells. CD86 is expressed at low levels in macrophages and high levels in dendritic cells. Therefore, we have tested the hypothesis that CD34(+)/CD86(+) cells are the common precursors of both macrophages and dendritic cells. CD34(+)/CD86(+) marrow cells cultured in granulocyte-macrophage colony-stimulating factor (GM-CSF)-generated macrophages. In contrast, CD34(+)/CD86(-) cells cultured in GM-CSF generated a predominant population of granulocytes. CD34(+)/CD86(+) cells cultured in GM-CSF plus tumor necrosis factor-alpha (TNF-alpha) generated almost exclusively CD1a+/CD83(+) dendritic cells. In contrast, CD34(+)/CD86(-) cells cultured in GM-CSF plus TNF-alpha generated a variety of cell types, including a small population of dendritic cells. In addition, CD34(+)/CD86(+) cells cultured in granulocyte colony-stimulating factor failed to generate CD15(+) granulocytes. Therefore, CD34(+)/CD86(+) cells are committed precursors of both macrophages and dendritic cells. The ontogeny of dendritic cells was recapitulated by stimulation of CD34(+)/CD86(-) cells with TNF-alpha that induced expression of CD86. Subsequent costimulation of CD86(+) cells with GM-CSF plus TNF-alpha lead to expression of CD83 and produced terminal dendritic cell differentiation. Thus, expression of CD86 on hematopoietic progenitor cells is regulated by TNF-alpha and denotes differentiation towards the macrophage or dendritic cell lineages.

  11. Higher Vulnerability and Stress Sensitivity of Neuronal Precursor Cells Carrying an Alpha-Synuclein Gene Triplication

    PubMed Central

    Flierl, Adrian; Oliveira, Luís M. A.; Falomir-Lockhart, Lisandro J.; Mak, Sally K.; Hesley, Jayne; Soldner, Frank; Arndt-Jovin, Donna J.; Jaenisch, Rudolf; Langston, J. William; Jovin, Thomas M.; Schüle, Birgitt

    2014-01-01

    Parkinson disease (PD) is a multi-factorial neurodegenerative disorder with loss of dopaminergic neurons in the substantia nigra and characteristic intracellular inclusions, called Lewy bodies. Genetic predisposition, such as point mutations and copy number variants of the SNCA gene locus can cause very similar PD-like neurodegeneration. The impact of altered α-synuclein protein expression on integrity and developmental potential of neuronal stem cells is largely unexplored, but may have wide ranging implications for PD manifestation and disease progression. Here, we investigated if induced pluripotent stem cell-derived neuronal precursor cells (NPCs) from a patient with Parkinson’s disease carrying a genomic triplication of the SNCA gene (SNCA-Tri). Our goal was to determine if these cells these neuronal precursor cells already display pathological changes and impaired cellular function that would likely predispose them when differentiated to neurodegeneration. To achieve this aim, we assessed viability and cellular physiology in human SNCA-Tri NPCs both under normal and environmentally stressed conditions to model in vitro gene-environment interactions which may play a role in the initiation and progression of PD. Human SNCA-Tri NPCs displayed overall normal cellular and mitochondrial morphology, but showed substantial changes in growth, viability, cellular energy metabolism and stress resistance especially when challenged by starvation or toxicant challenge. Knockdown of α-synuclein in the SNCA-Tri NPCs by stably expressed short hairpin RNA (shRNA) resulted in reversal of the observed phenotypic changes. These data show for the first time that genetic alterations such as the SNCA gene triplication set the stage for decreased developmental fitness, accelerated aging, and increased neuronal cell loss. The observation of this “stem cell pathology” could have a great impact on both quality and quantity of neuronal networks and could provide a powerful new

  12. CCR2 mediates the uptake of bone marrow-derived fibroblast precursors in angiotensin II-induced cardiac fibrosis

    PubMed Central

    Xu, Jing; Lin, Song-Chang; Chen, Jiyuan; Miao, Yuanxin; Taffet, George E.; Entman, Mark L.

    2011-01-01

    Angiotensin II plays an important role in the development of cardiac hypertrophy and fibrosis, but the underlying cellular and molecular mechanisms are not completely understood. Recent studies have shown that bone marrow-derived fibroblast precursors are involved in the pathogenesis of cardiac fibrosis. Since bone marrow-derived fibroblast precursors express chemokine receptor, CCR2, we tested the hypothesis that CCR2 mediates the recruitment of fibroblast precursors into the heart, causing angiotensin II-induced cardiac fibrosis. Wild-type and CCR2 knockout mice were infused with angiotensin II at 1,500 ng·kg−1·min−1. Angiotensin II treatment resulted in elevated blood pressure and cardiac hypertrophy that were not significantly different between wild-type and CCR2 knockout mice. Angiotensin II treatment of wild-type mice caused prominent cardiac fibrosis and accumulation of bone marrow-derived fibroblast precursors expressing the hematopoietic markers, CD34 and CD45, and the mesenchymal marker, collagen I. However, angiotensin II-induced cardiac fibrosis and accumulation of bone marrow-derived fibroblast precursors in the heart were abrogated in CCR2 knockout mice. Furthermore, angiotensin II treatment of wild-type mice increased the levels of collagen I, fibronectin, and α-smooth muscle actin in the heart, whereas these changes were not observed in the heart of angiotensin II-treated CCR2 knockout mice. Functional studies revealed that the reduction of cardiac fibrosis led to an impairment of cardiac systolic function and left ventricular dilatation in angiotensin II-treated CCR2 knockout mice. Our data demonstrate that CCR2 plays a pivotal role in the pathogenesis of angiotensin II-induced cardiac fibrosis through regulation of bone marrow-derived fibroblast precursors. PMID:21572015

  13. Unfolding biological properties of a versatile dicopper(II) precursor and its two mononuclear copper(II) derivatives.

    PubMed

    Paul, Anup; Hazra, Susanta; Sharma, Gunjan; Guedes da Silva, M Fátima C; Koch, Biplob; Pombeiro, Armando J L

    2017-09-01

    Synthesis, inter-conversions and biological study of the dichloro bridged dicopper(II) compound [CuLCl]2 (1) and its two mononuclear derivatives [CuLCl(H2O)]·H2O (2) and [CuLCl(py)] (3) (HL=2-(2-pyridylmethyleneamino)benzenesulfonic acid) are described. The dimeric compound 1 collapses into monomers 2 and 3 in the presence of coordinating solvents, water and pyridine, respectively, and 1 is regenerated upon simple stirring of 2 or 3 in methanol. The reactions of 1 with neutral (present study) and charged (earlier studies) ligands result in monomeric and multimeric compounds, respectively, attesting that it is a versatile dicopper(II) precursor. The anticancer activity of these copper complexes (1-3) was screened against lung (A-549) and breast (MDA-MB-231) human cancer cell lines. The IC50 (half maximal inhibitory concentration) value for one (3) of the compounds suggests preferential cytotoxicity against breast cancer MDA-MB-231 cell line. Furthermore, the IC50 value obtained for complex 3 is found to be almost two-fold times cytotoxic than the standard drug cisplatin. In addition, the underlying possible mechanism of its apoptosis-inducing efficacy in MDA-MB-231 cells has been rationalized by using flow cytometry (FACS) and Hoechst 33342/propidium iodide (PI) fluorescence staining. The stimulation of apoptotic induction for complex 3 has further been affirmed by reactive oxygen species (ROS) generation and mitochondrial aggregations studies. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Neural Precursor-Derived Pleiotrophin Mediates Subventricular Zone Invasion by Glioma.

    PubMed

    Qin, Elizabeth Y; Cooper, Dominique D; Abbott, Keene L; Lennon, James; Nagaraja, Surya; Mackay, Alan; Jones, Chris; Vogel, Hannes; Jackson, Peter K; Monje, Michelle

    2017-08-24

    The lateral ventricle subventricular zone (SVZ) is a frequent and consequential site of pediatric and adult glioma spread, but the cellular and molecular mechanisms mediating this are poorly understood. We demonstrate that neural precursor cell (NPC):glioma cell communication underpins this propensity of glioma to colonize the SVZ through secretion of chemoattractant signals toward which glioma cells home. Biochemical, proteomic, and functional analyses of SVZ NPC-secreted factors revealed the neurite outgrowth-promoting factor pleiotrophin, along with required binding partners SPARC/SPARCL1 and HSP90B, as key mediators of this chemoattractant effect. Pleiotrophin expression is strongly enriched in the SVZ, and pleiotrophin knock down starkly reduced glioma invasion of the SVZ in the murine brain. Pleiotrophin, in complex with the binding partners, activated glioma Rho/ROCK signaling, and ROCK inhibition decreased invasion toward SVZ NPC-secreted factors. These findings demonstrate a pathogenic role for NPC:glioma interactions and potential therapeutic targets to limit glioma invasion. PAPERCLIP. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Reversal of developmental restrictions in neural crest lineages: Transition from Schwann cells to glial-melanocytic precursors in vitro

    PubMed Central

    Dupin, Elisabeth; Real, Carla; Glavieux-Pardanaud, Corinne; Vaigot, Pierre; Le Douarin, Nicole M.

    2003-01-01

    In vertebrate embryos, diversification of the lineages arising from the neural crest (NC) is controlled to a large extent by environmental factors. In previous work, we showed that endothelin 3 (ET3) peptide favors the development of glial and melanocytic NC precursors in vitro. This factor is also capable of inducing proliferation of cultured epidermal pigment cells and their conversion to glia. ET3 therefore strongly promotes the emergence of melanocytic and glial phenotypes from precursors and acts on the maintenance of these phenotypes. In the present work, we explored the capacity of ET3 to reprogram glial cells into melanocytes. Schwann cells expressing glial-specific markers [such as the Schwann cell myelin protein (SMP)] were isolated from sciatic nerves of quail embryos and cultured in vitro. We found that ET3 promotes cell growth and sequential expression of melanocyte differentiation markers in cultures of purified SMP-expressing cells, whereas it had no significant effect on SMP-negative cells from the same nerves. Moreover, we provide evidence for the transition of differentiated Schwann cells to melanocytes in clonal cultures. This transition involves the production of a mixed progeny of melanoblasts/melanocytes, glia, and cells bearing differentiation markers of both phenotypes. Therefore, Schwann cells exposed to ET3 transdifferentiate to melanocytes through reversion to the stage of bipotent glial-melanocytic NC precursors. These findings show that NC-derived pigment and glial cells are phenotypically unstable in vitro and may undergo reversal of precursor hierarchy to function as bipotent stem cells. PMID:12702775

  16. Concomitant T-cell receptor alpha and delta gene rearrangements in individual T-cell precursors.

    PubMed Central

    Thompson, S D; Pelkonen, J; Hurwitz, J L

    1990-01-01

    A debate has recently surfaced concerning the degree of precommitment attained by alpha beta and gamma delta T-cell precursors prior to T-cell receptor (TCR) gene rearrangement. It has been suggested that precursors may be precommitted to rearrange either alpha or delta genes, but not both, thus giving rise to alpha beta- and gamma delta-producing T cells, respectively. Alternatively, the precursors may be flexible with regard to potential TCR gene rearrangements. To address this controversy, the gene rearrangements among a group of T-cell hybridomas from fetal, newborn, and early postnatal mouse thymi were examined. Six probes spanning the delta and alpha loci were used in Southern blot analyses to characterize the rearrangements which occurred on homologous chromosomes in each cell. Although homologous chromosomes often rearranged in synchrony within the alpha locus, a number of hybridomas were found which had retained a delta rearrangement on one chromosome and an alpha rearrangement on the second. Results show that a precommitment by T cells to rearrange delta or alpha genes in a mutually exclusive manner is not an absolute feature of mouse thymocyte development. Images PMID:2164690

  17. Triazole RGD antagonist reverts TGFβ1-induced endothelial-to-mesenchymal transition in endothelial precursor cells.

    PubMed

    Bianchini, Francesca; Peppicelli, Silvia; Fabbrizzi, Pierangelo; Biagioni, Alessio; Mazzanti, Benedetta; Menchi, Gloria; Calorini, Lido; Pupi, Alberto; Trabocchi, Andrea

    2017-01-01

    Fibrosis is the dramatic consequence of a dysregulated reparative process in which activated fibroblasts (myofibroblasts) and Transforming Growth Factor β1 (TGFβ1) play a central role. When exposed to TGFβ1, fibroblast and epithelial cells differentiate in myofibroblasts; in addition, endothelial cells may undergo endothelial-to-mesenchymal transition (EndoMT) and actively participate to the progression of fibrosis. Recently, the role of αv integrins, which recognize the Arg-Gly-Asp (RGD) tripeptide, in the release and signal transduction activation of TGFβ1 became evident. In this study, we present a class of triazole-derived RGD antagonists that interact with αvβ3 integrin. Above different compounds, the RGD-2 specifically interferes with integrin-dependent TGFβ1 EndoMT in Endothelial Colony-Forming Cells (ECPCs) derived from circulating Endothelial Precursor Cells (ECPCs). The RGD-2 decreases the amount of membrane-associated TGFβ1, and reduces both ALK5/TGFβ1 type I receptor expression and Smad2 phosphorylation in ECPCs. We found that RGD-2 antagonist reverts EndoMT, reducing α-smooth muscle actin (α-SMA) and vimentin expression in differentiated ECPCs. Our results outline the critical role of integrin in fibrosis progression and account for the opportunity of using integrins as target for anti-fibrotic therapeutic treatment.

  18. T cell dysfunction in the diabetes-prone BB rat. A role for thymic migrants that are not T cell precursors

    SciTech Connect

    Georgiou, H.M.; Lagarde, A.C.; Bellgrau, D.

    1988-01-01

    Diabetes-prone BB (BB-DP) rats express several T cell dysfunctions which include poor proliferative and cytotoxic responses to alloantigen. The goal of this study was to determine the origin of these T cell dysfunctions. When BB-DP rats were thymectomized, T cell depleted, and transplanted with neonatal thymus tissue from diabetes-resistant and otherwise normal DA/BB F1 rats, the early restoration of T cell function proceeded normally on a cell-for-cell basis; i.e., peripheral T cells functioned like those from the thymus donor. Because the thymus in these experiments was subjected to gamma irradiation before transplantation and there was no evidence of F1 chimerism in the transplanted BB-DP rats, it appeared that the BB-DP T cell precursors could mature into normally functioning T cells if the maturation process occurred in a normal thymus. If the F1 thymus tissue was treated with dGua before transplantation, the T cells of these animals functioned poorly like those from untreated BB-DP rats. dGua poisons bone marrow-derived cells, including gamma radiation-resistant cells of the macrophage/dendritic cell lineages, while sparing the thymic epithelium. Therefore, the reversal of the T cell dysfunction depends on the presence in the F1 thymus of gamma radiation-resistant, dGua-sensitive F1 cells. Conversely, thymectomized and T cell-depleted F1 rats expressed T cell dysfunction when transplanted with gamma-irradiated BB thymus grafts. T cell responses were normal in animals transplanted with dGua-treated BB thymus grafts. With increasing time after thymus transplantation, T cells from all animals gradually expressed the functional phenotype of the bone marrow donor. Taken together these results suggest that BB-DP bone marrow-derived cells that are not T cell precursors influence the maturation environment in the thymus of otherwise normal BB-DP T cell precursors.

  19. Carbon-Nanodot Solar Cells from Renewable Precursors.

    PubMed

    Marinovic, Adam; Kiat, Lim S; Dunn, Steve; Titirici, Maria-Magdalena; Briscoe, Joe

    2017-03-09

    It has recently been shown that waste biomass can be converted into a wide range of functional materials, including those with desirable optical and electronic properties, offering the opportunity to find new uses for these renewable resources. Photovoltaics is one area in which finding the combination of abundant, low-cost and non-toxic materials with the necessary functionality can be challenging. In this paper the performance of carbon nanodots derived from a wide range of biomaterials obtained from different biomass sources as sensitisers for TiO2 -based nanostructured solar cells was compared; polysaccharides (chitosan and chitin), monosaccharide (d-glucose), amino acids (l-arginine and l-cysteine) and raw lobster shells were used to produce carbon nanodots through hydrothermal carbonisation. The highest solar power conversion efficiency (PCE) of 0.36 % was obtained by using l-arginine carbon nanodots as sensitisers, whereas lobster shells, as a model source of chitin from actual food waste, showed a PCE of 0.22 %. By comparing this wide range of materials, the performance of the solar cells was correlated with the materials characteristics by carefully investigating the structural and optical properties of each family of carbon nanodots, and it was shown that the combination of amine and carboxylic acid functionalisation is particularly beneficial for the solar-cell performance.

  20. Perspectives on the role of Pannexin 1 in neural precursor cell biology.

    PubMed

    Sanchez-Arias, Juan C; Wicki-Stordeur, Leigh E; Swayne, Leigh Anne

    2016-10-01

    We recently reported that targeted deletion of Pannexin 1 in neural precursor cells of the ventricular zone impairs the maintenance of these cells in healthy and stroke-injured brain. Here we frame this exciting new finding in the context of our previous studies on Pannexin 1 in neural precursors as well as the close relationship between Pannexin 1 and purinergic receptors established by other groups. Moreover, we identify important gaps in our understanding of Pannexin 1 in neural precursor cell biology in terms of the underlying molecular mechanisms and functional/behavioural outcomes.

  1. Perspectives on the role of Pannexin 1 in neural precursor cell biology

    PubMed Central

    Sanchez-Arias, Juan C.; Wicki-Stordeur, Leigh E.; Swayne, Leigh Anne

    2016-01-01

    We recently reported that targeted deletion of Pannexin 1 in neural precursor cells of the ventricular zone impairs the maintenance of these cells in healthy and stroke-injured brain. Here we frame this exciting new finding in the context of our previous studies on Pannexin 1 in neural precursors as well as the close relationship between Pannexin 1 and purinergic receptors established by other groups. Moreover, we identify important gaps in our understanding of Pannexin 1 in neural precursor cell biology in terms of the underlying molecular mechanisms and functional/behavioural outcomes. PMID:27904473

  2. Optical and mechanical behaviors of glassy silicone networks derived from linear siloxane precursors

    NASA Astrophysics Data System (ADS)

    Jang, Heejun; Seo, Wooram; Kim, Hyungsun; Lee, Yoonjoo; Kim, Younghee

    2016-01-01

    Silicon-based inorganic polymers are promising materials as matrix materials for glass fiber composites because of their good process ability, transparency, and thermal property. In this study, for utilization as a matrix precursor for a glass-fiber-reinforced composite, glassy silicone networks were prepared via hydrosilylation of linear/pendant Si-H polysiloxanes and the C=C bonds of viny-lterminated linear/cyclic polysiloxanes. 13C nuclear magnetic resonance spectroscopy was used to determine the structure of the cross-linked states, and a thermal analysis was performed. To assess the mechanical properties of the glassy silicone networks, we performed nanoindentation and 4-point bending tests. Cross-linked networks derived from siloxane polymers are thermally and optically more stable at high temperatures. Different cross-linking agents led to final networks with different properties due to differences in the molecular weights and structures. After stepped postcuring, the Young's modulus and the hardness of the glassy silicone networks increased; however, the brittleness also increased. The characteristics of the cross-linking agent played an important role in the functional glassy silicone networks.

  3. [Antitoxic properties of pantothenic acid derivatives, precursors of coenzyme A biosynthesis, with regard to kanamycin].

    PubMed

    Moĭseenok, A G; Dorofeev, B F; Sheĭbak, V M; Khomich, T I

    1984-11-01

    The effect of calcium pantothenate (CPN)B 4'-phospho-CPN (PCP), pantetheine (PT) and calcium S-sulfopantetheine (SPN) on acute toxicity of kanamycin sulfate was studied on albino mice. The above derivatives of pantothenic acid except PT lowered the antibiotic toxicity. The coefficient of the antitoxic effect (LD50/ED50) of SPN and PCP was 1.3-1.4 times higher than that of CPN. The combined use of kanamycin (1/5 of the LD50) with CPN, PCP or PT (30 mg/kg bw was equivalent to CPN) for 15 days prevented the increase in the total content of CoA and in the content of the fraction of free CoA and the precursors of its biosynthesis participating in the reaction of N-acetylation in the liver and brain. The contents of these substances were within the normal during the whole experiment. A certain increase in the activity of pantothenate kinase in the liver cytosol due to the use of kanamycin was eliminated by the simultaneous use of PCP and PT. The vitamin-containing compounds PCP and SPN were recommended for the clinical trials as agents preventing complications of kanamycin therapy.

  4. Soot aggregate restructuring due to coatings of secondary organic aerosol derived from aromatic precursors.

    PubMed

    Schnitzler, Elijah G; Dutt, Ashneil; Charbonneau, André M; Olfert, Jason S; Jäger, Wolfgang

    2014-12-16

    Restructuring of monodisperse soot aggregates due to coatings of secondary organic aerosol (SOA) derived from hydroxyl radical-initiated oxidation of toluene, p-xylene, ethylbenzene, and benzene was investigated in a series of photo-oxidation (smog) chamber experiments. Soot aggregates were generated by combustion of ethylene using a McKenna burner, treated by denuding, size-selected by a differential mobility analyzer, and injected into a smog chamber, where they were exposed to low vapor pressure products of aromatic hydrocarbon oxidation, which formed SOA coatings. Aggregate restructuring began once a threshold coating mass was reached, and the degree of the subsequent restructuring increased with mass growth factor. Although significantly compacted, fully processed aggregates were not spherical, with a mass-mobility exponent of 2.78, so additional SOA was required to fill indentations between collapsed branches of the restructured aggregates before the dynamic shape factor of coated particles approached 1. Trends in diameter growth factor, effective density, and dynamic shape factor with increasing mass growth factor indicate distinct stages in soot aggregate processing by SOA coatings. The final degree and coating mass dependence of soot restructuring were found to be the same for SOA coatings from all four aromatic precursors, indicating that the surface tensions of the SOA coatings are similar.

  5. CD4+ T cell anergy prevents autoimmunity and generates regulatory T cell precursors

    PubMed Central

    Kalekar, Lokesh A.; Schmiel, Shirdi E.; Nandiwada, Sarada L.; Lam, Wing Y.; Barsness, Laura O.; Zhang, Na; Stritesky, Gretta L.; Malhotra, Deepali; Pauken, Kristen E.; Linehan, Jonathan L.; O’Sullivan, M. Gerard; Fife, Brian T.; Hogquist, Kristin A.; Jenkins, Marc K.; Mueller, Daniel L.

    2015-01-01

    The role that anergy, an acquired state of T cell functional unresponsiveness, plays in natural peripheral tolerance remains unclear. In this study, we demonstrate that anergy is selectively induced in fetal antigen-specific maternal CD4+ T cells during pregnancy. A naturally occurring subpopulation of anergic polyclonal CD4+ T cells, enriched in self antigen-specific T cell receptors, is also observed in healthy hosts. Neuropilin-1 expression in anergic conventional CD4+ T cells is associated with thymic regulatory T cell (Treg cell)-related gene hypomethylation, and this correlates with their capacity to differentiate into Foxp3+ Treg cells that suppress immunopathology. Thus, our data suggest that not only is anergy induction important in preventing autoimmunity, but it also generates the precursors for peripheral Treg cell differentiation. PMID:26829766

  6. Monocyte Heterogeneity: Consequences for Monocyte-Derived Immune Cells.

    PubMed

    Sprangers, Sara; de Vries, Teun J; Everts, Vincent

    2016-01-01

    Blood monocytes are precursors of dendritic cells, macrophages, and osteoclasts. They are a heterogeneous cell population with differences in size, phenotype, and function. Although monocytes maintain several tissue-specific populations of immune cells in homeostasis, their contribution to populations of dendritic cells, macrophages, and osteoclasts is significantly increased in inflammation. Identification of a growing number of functionally different subsets of cells within populations of monocyte-derived immune cells has recently put monocyte heterogeneity into sharp focus. Here, we summarize recent findings in monocyte heterogeneity and their differentiation into dendritic cells, macrophages, and osteoclasts. We also discuss these advances in the context of the formation of functionally different monocyte-derived subsets of dendritic cells, macrophages, and osteoclasts.

  7. Mouse fetal and embryonic liver cells differentiate human umbilical cord blood (UCB) progenitors into CD56 negative NK cell precursors in the absence of IL-15

    PubMed Central

    McCullar, Valarie; Oostendorp, Robert; Panoskaltsis-Mortari, Angela; Yung, Gong; Lutz, Charles T.; Wagner, John E.; Miller, Jeffrey S.

    2008-01-01

    Objective Human NK cell maturation involves the orderly acquisition of NK cell receptors. Our aim was to understand how stromal interactions and cytokines are important in this developmental process. Methods Human UCB CD34+/Lin−/CD38− cells were cultured on two murine stromal cell lines (AFT024 and EL08-1D2) in a switch culture to study NK cell development. Results When human progenitors were cultured on AFT024 with IL-3 and Flt3-L in the absence of IL-15, NK cell differentiation occurred, albeit at low frequency. These conditions favored the accumulation of CD56− NK cell precursors (CD34+CD7−, CD34+CD7+ and CD34−CD7+ cells), which are populations rare in adult blood but abundant in fresh UCB. In secondary culture, addition of IL-3 or IL-3+Flt3L to IL-15 increased the absolute number of CD56+ NK cells from precursors and the acquisition of CD94 and KIR. To further explore the microenvironment in early NK cell maturation, a cell line derived from murine embryonic liver (EL08-1D2) was studied. NK cell development and KIR acquisition was superior with EL08-1D2 which supported the differentiation of NK cell precursors, NK cell commitment, and proliferation. Conclusion Although the earliest events in NK cell maturation do not require exogenous human IL-15, it is required at a later stage of NK cell commitment. At a minimum, murine stroma, IL-3, and Flt3-L are required to recapitulate early NK cell development and differentiation into distinct NK cell precursors. EL08-1D2 induces KIR acquisition suggesting that extrinsic signals in NK cell development are conserved between mouse and man. PMID:18295962

  8. [Exosomes derived from dendritic cells].

    PubMed

    Amigorena, S

    2001-01-01

    Dendritic cells (DC) are potent antigen presenting cells and the only ones capable of inducing primary cytotoxic immune responses both in vivo and vitro. DCs secrete a 60-80 nm membrane vesicle population of endocytic origin, called exosomes. The protein composition of exosomes was analyzed using a systematic proteomic approach. Besides MHC and costimulatory molecules, exosomes bear several adhesion proteins, probably involved in their specific targeting. Exosomes also accumulate several cytosolic factors, most likely involved in exoxome's biogenesis in late endosomes. Like DCs, exosomes induce potent anti tumor immune responses in vivo. Indeed, a single injection of DC-derived exosomes sensitized with tumor peptides induced the eradication of established mouse tumors. Tumor-specific cytotoxic T lymphocytes were found in the spleen of exosome treated mice, and depletion of CD8+ T cells in vivo inhibited the anti tumor effect of exosomes. These results strongly support the implementation of human DC-derived exosomes for cancer immunotherapy.

  9. An Atlas of Network Topologies Reveals Design Principles for Caenorhabditis elegans Vulval Precursor Cell Fate Patterning.

    PubMed

    Ping, Xianfeng; Tang, Chao

    2015-01-01

    The vulval precursor cell (VPC) fate patterning in Caenorhabditis elegans is a classic model experimental system for cell fate determination and patterning in development. Despite its apparent simplicity (six neighboring cells arranged in one dimension) and many experimental and computational efforts, the patterning strategy and mechanism remain controversial due to incomplete knowledge of the complex biology. Here, we carry out a comprehensive computational analysis and obtain a reservoir of all possible network topologies that are capable of VPC fate patterning under the simulation of various biological environments and regulatory rules. We identify three patterning strategies: sequential induction, morphogen gradient and lateral antagonism, depending on the features of the signal secreted from the anchor cell. The strategy of lateral antagonism, which has not been reported in previous studies of VPC patterning, employs a mutual inhibition of the 2° cell fate in neighboring cells. Robust topologies are built upon minimal topologies with basic patterning strategies and have more flexible and redundant implementations of modular functions. By simulated mutation, we find that all three strategies can reproduce experimental error patterns of mutants. We show that the topology derived by mapping currently known biochemical pathways to our model matches one of our identified functional topologies. Furthermore, our robustness analysis predicts a possible missing link related to the lateral antagonism strategy. Overall, we provide a theoretical atlas of all possible functional networks in varying environments, which may guide novel discoveries of the biological interactions in vulval development of Caenorhabditis elegans and related species.

  10. An Atlas of Network Topologies Reveals Design Principles for Caenorhabditis elegans Vulval Precursor Cell Fate Patterning

    PubMed Central

    Ping, Xianfeng; Tang, Chao

    2015-01-01

    The vulval precursor cell (VPC) fate patterning in Caenorhabditis elegans is a classic model experimental system for cell fate determination and patterning in development. Despite its apparent simplicity (six neighboring cells arranged in one dimension) and many experimental and computational efforts, the patterning strategy and mechanism remain controversial due to incomplete knowledge of the complex biology. Here, we carry out a comprehensive computational analysis and obtain a reservoir of all possible network topologies that are capable of VPC fate patterning under the simulation of various biological environments and regulatory rules. We identify three patterning strategies: sequential induction, morphogen gradient and lateral antagonism, depending on the features of the signal secreted from the anchor cell. The strategy of lateral antagonism, which has not been reported in previous studies of VPC patterning, employs a mutual inhibition of the 2° cell fate in neighboring cells. Robust topologies are built upon minimal topologies with basic patterning strategies and have more flexible and redundant implementations of modular functions. By simulated mutation, we find that all three strategies can reproduce experimental error patterns of mutants. We show that the topology derived by mapping currently known biochemical pathways to our model matches one of our identified functional topologies. Furthermore, our robustness analysis predicts a possible missing link related to the lateral antagonism strategy. Overall, we provide a theoretical atlas of all possible functional networks in varying environments, which may guide novel discoveries of the biological interactions in vulval development of Caenorhabditis elegans and related species. PMID:26114587

  11. Loss of T cell precursors after spaceflight and exposure to vector-averaged gravity

    NASA Technical Reports Server (NTRS)

    Woods, Chris C.; Banks, Krista E.; Gruener, Raphael; DeLuca, Dominick

    2003-01-01

    Using fetal thymus organ culture (FTOC), we examined the effects of spaceflight and vector-averaged gravity on T cell development. Under both conditions, the development of T cells was significantly attenuated. Exposure to spaceflight for 16 days resulted in a loss of precursors for CD4+, CD8+, and CD4+CD8+ T cells in a rat/mouse xenogeneic co-culture. A significant decrease in the same precursor cells, as well as a decrease in CD4-CD8- T cell precursors, was also observed in a murine C57BL/6 FTOC after rotation in a clinostat to produce a vector-averaged microgravity-like environment. The block in T cell development appeared to occur between the pre-T cell and CD4+CD8+ T cell stage. These data indicate that gravity plays a decisive role in the development of T cells.

  12. Loss of T cell precursors after spaceflight and exposure to vector-averaged gravity

    NASA Technical Reports Server (NTRS)

    Woods, Chris C.; Banks, Krista E.; Gruener, Raphael; DeLuca, Dominick

    2003-01-01

    Using fetal thymus organ culture (FTOC), we examined the effects of spaceflight and vector-averaged gravity on T cell development. Under both conditions, the development of T cells was significantly attenuated. Exposure to spaceflight for 16 days resulted in a loss of precursors for CD4+, CD8+, and CD4+CD8+ T cells in a rat/mouse xenogeneic co-culture. A significant decrease in the same precursor cells, as well as a decrease in CD4-CD8- T cell precursors, was also observed in a murine C57BL/6 FTOC after rotation in a clinostat to produce a vector-averaged microgravity-like environment. The block in T cell development appeared to occur between the pre-T cell and CD4+CD8+ T cell stage. These data indicate that gravity plays a decisive role in the development of T cells.

  13. Human post-thymic precursor cells in health and disease. I. Characterization of the autologous rosette-forming T cells as post-thymic precursors.

    PubMed Central

    Palacios, R; Alarcón-Segovia, D; Llorente, L; Ruíz-Arguelles, A; Díaz-Jouanen, E

    1981-01-01

    Human autologous-rosette-forming T cells (Tar cells) have many of the characteristics of post-thymic precursor cells. Thus, they bind to sheep erythrocytes but have neither receptors for the Fc portion of IgG nor for that of IgM. They include a subpopulation that binds peanut agglutinin which suggests that they are immature and, as opposed to T cells with either receptors for the FC portion of IgM (T mu) or of IgG (T gamma), Tar cells adhere to nylon wool, another possible indicator of immaturity, as is their extreme sensitivity to hydrocortisone both in vitro and in vivo. There are more Tar cells in cord blood than in the peripheral blood of young adults and there are more Tar cells in the peripheral blood of young adults than in the peripheral blood of elderly subjects. By co-culturing T mu and B cells, or T mu, or Tar and B cells in the presence of pokeweek mitogen (PWM) we were able to determine that these cells cause feedback inhibition, a function considered characteristic of post-thymic precursors. In co-cultures in which we placed mononuclear cells (MNC) or MNC plus Tar cells, or MNC depleted of Tar cells or MNC depleted of Tar cells plus Tar cells stimulated with PWM, we determined that Tar cells play a role in the generation of suppression thereby confirming that human Tar cells are precursor cells. We also found that Tar cells proliferated and generated T gamma and T mu cells both spontaneously and in greater numbers, under the effect of serum thymic factor. PMID:6970170

  14. Hedgehog signaling establishes precursors for germline stem cell niches by regulating cell adhesion

    PubMed Central

    Lin, Kun-Yang; Kao, Shih-Han

    2017-01-01

    Stem cells require different types of supporting cells, or niches, to control stem cell maintenance and differentiation. However, little is known about how those niches are formed. We report that in the development of the Drosophila melanogaster ovary, the Hedgehog (Hh) gradient sets differential cell affinity for somatic gonadal precursors to specify stromal intermingled cells, which contributes to both germline stem cell maintenance and differentiation niches in the adult. We also report that Traffic Jam (an orthologue of a large Maf transcription factor in mammals) is a novel transcriptional target of Hh signaling to control cell–cell adhesion by negative regulation of E-cadherin expression. Our results demonstrate the role of Hh signaling in niche establishment by segregating somatic cell lineages for differentiation. PMID:28363970

  15. Induction of cytotoxic T cell precursors in vivo. Role of T helper cells

    PubMed Central

    1983-01-01

    Strain AS rats respond with two populations of cytotoxic T lymphocytes to stimulation in vitro by the major histocompatibility complex (MHC)- incompatible strain HL rat tumor (HL-A2T2). One is specific for MHC alloantigens present on both HL-A2T2 and normal HL targets, the other is tumor specific. The activation of these killer cells requires helper T lymphocytes. The tumor-specific helper cells depend on syngeneic radioresistant accessory cells to present the tumor antigens in an immunogenic form. The appropriate helper-accessory cell interaction results in the production of soluble factors which then induce the maturation of precursor cells into effective killer cells. Studies with a procedure for inducing negative selection of T cells in vivo showed that short-term exposure to HL-A2T2 tumor induced selection only for TH but not cytotoxic T lymphocyte precursors (CTLp). Simultaneous injection of supernatants from concanavalin A-activated spleen cell cultures, however, did produce selection of CTLp. These and other findings suggest that under normal circumstances in vivo, both signals (recognition of antigen and acceptance of maturation factors) are provided in the vicinity of an antigen presenting macrophage-like accessory cell. PMID:6222131

  16. Inducible T-cell receptor expression in precursor T-cells for leukemia control

    PubMed Central

    Hoseini, Shahabuddin S; Hapke, Martin; Herbst, Jessica; Wedekind, Dirk; Baumann, Rolf; Heinz, Niels; Schiedlmeier, Bernhard; Vignali, Dario AA; van den Brink, Marcel R.M.; Schambach, Axel; Blazar, Bruce R.; Sauer, Martin G.

    2015-01-01

    Co-transplantation of hematopoietic stem cells with those engineered to express leukemia-reactive T cell receptors (TCRs) and differentiated ex vivo into precursor T cells (preTs) may reduce the risk of leukemia relapse. Since expression of potentially self-(leukemia-) reactive TCRs will lead to negative selection or provoke autoimmunity upon thymic maturation, we investigated a novel concept whereby TCR expression set under the control of an inducible promoter would allow timely controlled TCR expression. After in vivo maturation and gene induction, preTs developed potent anti-leukemia effects. Engineered preTs provided protection even after repeated leukemia challenges by giving rise to effector and central memory cells. Importantly, adoptive transfer of TCR-transduced allogeneic preTs mediated anti-leukemia effect without evoking graft-versus-host disease (GVHD). Earlier transgene induction forced CD8+ T cell development, was required to obtain a mature T cell subset of targeted specificity, allowed engineered T cells to efficiently pass positive selection and abrogated the endogenous T cell repertoire. Later induction favored CD4 differentiation and failed to produce a leukemia-reactive population emphasizing the dominant role of positive selection. Taken together, we provide new functional insights for the employment of TCR-engineered precursor cells as a controllable immunotherapeutic modality with significant anti-leukemia activity. PMID:25652739

  17. Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia.

    PubMed

    Haso, Waleed; Lee, Daniel W; Shah, Nirali N; Stetler-Stevenson, Maryalice; Yuan, Constance M; Pastan, Ira H; Dimitrov, Dimiter S; Morgan, Richard A; FitzGerald, David J; Barrett, David M; Wayne, Alan S; Mackall, Crystal L; Orentas, Rimas J

    2013-02-14

    Immune targeting of B-cell malignancies using chimeric antigen receptors (CARs) is a promising new approach, but critical factors impacting CAR efficacy remain unclear. To test the suitability of targeting CD22 on precursor B-cell acute lymphoblastic leukemia (BCP-ALL), lymphoblasts from 111 patients with BCP-ALL were assayed for CD22 expression and all were found to be CD22-positive, with median CD22 expression levels of 3500 sites/cell. Three distinct binding domains targeting CD22 were fused to various TCR signaling domains ± an IgG heavy chain constant domain (CH2CH3) to create a series of vector constructs suitable to delineate optimal CAR configuration. CARs derived from the m971 anti-CD22 mAb, which targets a proximal CD22 epitope demonstrated superior antileukemic activity compared with those incorporating other binding domains, and addition of a 4-1BB signaling domain to CD28.CD3 constructs diminished potency, whereas increasing affinity of the anti-CD22 binding motif, and extending the CD22 binding domain away from the membrane via CH2CH3 had no effect. We conclude that second-generation m971 mAb-derived anti-CD22 CARs are promising novel therapeutics that should be tested in BCP-ALL.

  18. Adult neurogenesis in the crayfish brain: proliferation, migration and possible origin of precursor cells

    PubMed Central

    Zhang, Y.; Allodi, S.; Sandeman, D.C.; Beltz, B.S.

    2015-01-01

    The birth of new neurons and their incorporation into functional circuits in the adult brain is a characteristic of many vertebrate and invertebrate organisms, including decapod crustaceans. Precursor cells maintaining life-long proliferation in the brains of crayfish (Procambarus clarkii, Cherax destructor) and clawed lobsters (Homarus americanus) reside within a specialized niche on the ventral surface of the brain; their daughters migrate to two proliferation zones along a stream formed by processes of the niche precursors. Here they divide again, finally producing interneurons in the olfactory pathway. The present studies in P. clarkii explore (1) differential proliferative activity among the niche precursor cells with growth and aging, (2) morphological characteristics of cells in the niche and migratory streams, and (3) aspects of the cell cycle in this lineage. Morphologically symmetrical divisions of neuronal precursor cells were observed in the niche near where the migratory streams emerge, as well as in the streams and proliferation zones. The nuclei of migrating cells elongate and undergo shape changes consistent with nucleokinetic movement. LIS1, a highly conserved dynein-binding protein, is expressed in cells in the migratory stream and neurogenic niche, implicating this protein in the translocation of crustacean brain neuronal precursor cells. Symmetrical divisions of the niche precursors and migration of both daughters raised the question of how the niche precursor pool is replenished. We present here preliminary evidence for an association between vascular cells and the niche precursors, which may relate to the life-long growth and maintenance of the crustacean neurogenic niche. PMID:19294644

  19. Gene expression analysis of embryonic photoreceptor precursor cells using BAC-Crx-EGFP transgenic mouse.

    PubMed

    Muranishi, Yuki; Sato, Shigeru; Inoue, Tatsuya; Ueno, Shinji; Koyasu, Toshiyuki; Kondo, Mineo; Furukawa, Takahisa

    2010-02-12

    Crx is a transcription factor which is predominantly expressed in developing and mature photoreceptor cells in the retina, and plays a crucial role in the terminal differentiation of both rods and cones. Crx is one of the earliest-expressed genes specifically in photoreceptor precursors, allowing us to trace photoreceptor precursor cells from embryonic stages to adult stage by visualizing Crx-expressing cells. In the current study, we generated a transgenic mouse line which expresses enhanced green fluorescence protein (EGFP) in the retina driven by the Crx promoter using bacterial artificial chromosome (BAC) transgenesis. EGFP-positive cells were observed in the presumptive photoreceptor layer in the retina at embryonic day 15.5 (E15.5), and continued to be expressed in developing and mature photoreceptor cells up to adult stage. We sorted EGFP-positive photoreceptor precursors at E17.5 using fluorescence-activated cell sorter (FACS), and subsequently performed microarray analysis of the FACS-sorted cells. We observed various photoreceptor genes, especially cone genes, are enriched in the EGFP-positive cells, indicating that embryonic cone photoreceptor precursors are enriched. In addition, we found that most of the EGFP-positive cells were post-mitotic cells. Thus, the transgenic line we established can serve as a useful tool to study both developing and mature photoreceptor cells, including embryonic cone precursors whose analysis has been difficult.

  20. Human Neural Precursor Cells Promote Neurologic Recovery in a Viral Model of Multiple Sclerosis

    PubMed Central

    Chen, Lu; Coleman, Ronald; Leang, Ronika; Tran, Ha; Kopf, Alexandra; Walsh, Craig M.; Sears-Kraxberger, Ilse; Steward, Oswald; Macklin, Wendy B.; Loring, Jeanne F.; Lane, Thomas E.

    2014-01-01

    Summary Using a viral model of the demyelinating disease multiple sclerosis (MS), we show that intraspinal transplantation of human embryonic stem cell-derived neural precursor cells (hNPCs) results in sustained clinical recovery, although hNPCs were not detectable beyond day 8 posttransplantation. Improved motor skills were associated with a reduction in neuroinflammation, decreased demyelination, and enhanced remyelination. Evidence indicates that the reduced neuroinflammation is correlated with an increased number of CD4+CD25+FOXP3+ regulatory T cells (Tregs) within the spinal cords. Coculture of hNPCs with activated T cells resulted in reduced T cell proliferation and increased Treg numbers. The hNPCs acted, in part, through secretion of TGF-β1 and TGF-β2. These findings indicate that the transient presence of hNPCs transplanted in an animal model of MS has powerful immunomodulatory effects and mediates recovery. Further investigation of the restorative effects of hNPC transplantation may aid in the development of clinically relevant MS treatments. PMID:24936469

  1. Adult neural precursor cells from the subventricular zone contribute significantly to oligodendrocyte regeneration and remyelination.

    PubMed

    Xing, Yao Lulu; Röth, Philipp T; Stratton, Jo Anne S; Chuang, Bernard H A; Danne, Jill; Ellis, Sarah L; Ng, Sze Woei; Kilpatrick, Trevor J; Merson, Tobias D

    2014-10-15

    Parenchymal oligodendrocyte progenitor cells (pOPCs) are considered the principal cell type responsible for oligodendrogenesis and remyelinaton in demyelinating diseases. Recent studies have demonstrated that neural precursor cells (NPCs) from the adult subventricular zone (SVZ) can also generate new oligodendrocytes after demyelination. However, the relative contribution of NPCs versus pOPCs to remyelination is unknown. We used in vivo genetic fate mapping to assess the behavior of each progenitor type within the corpus callosi (CCs) of mice subjected to cuprizone-induced demyelination. Nestin-CreER(T2) and Pdgfra-CreER(T2) transgenic mice were crossed with fluorescent Cre reporter strains to map the fate of NPCs and pOPCs respectively. In cuprizone-challenged mice, substantial numbers of NPCs migrated into the demyelinated CC and contributed to oligodendrogenesis. This capacity was most prominent in rostral regions adjacent to the SVZ where NPC-derived oligodendrocytes significantly outnumbered those generated from pOPCs. Sixty-two percent of all nodes of Ranvier in this region were flanked by at least one paranode generated from an NPC-derived oligodendrocyte. Remarkably, g-ratios (ratio of the axon diameter to the diameter of the axon plus myelin sheath) of myelinated axons in regions subject to significant NPC-derived remyelination were equivalent to those of unchallenged controls, and immunoelectron microscopy revealed that NPC-derived myelin was significantly thicker than that generated by pOPCs, regardless of axonal caliber. We also demonstrate that a reduced efficiency of remyelination in the caudal CC was associated with long-term impairment in the maturation of oligodendrogenic NPCs but only transient delay in pOPC differentiation. Collectively, our data define a major distinct role for NPCs in remyelination, identifying them as a key target for enhancing myelin repair in demyelinating diseases.

  2. Characterization of the anti-CD22 targeted therapy, moxetumomab pasudotox, for B-cell precursor acute lymphoblastic leukemia.

    PubMed

    Kinjyo, Ichiko; Matlawska-Wasowska, Ksenia; Chen, Xiaoru; Monks, Noel R; Burke, Patricia; Winter, Stuart S; Wilson, Bridget S

    2017-11-01

    Moxetumomab pasudotox is a second-generation recombinant immunotoxin against CD22 on B-cell lineages. Antileukemic activity has been demonstrated in children with chemotherapy-refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL), with variable responses. Here, we report in vitro and in vivo evaluation of moxetumomab pasudotox treatment of human cell lines and patient-derived cells as a preliminary study to understand characteristics of sensitivity to treatment. Binding, internalization, and apoptosis were evaluated using fluorescently tagged moxetumomab pasudotox. Studies in NOD-scid IL2Rg(null) mice showed a modest survival benefit in mice engrafted with 697 cells but not in NALM6 or the two patient-derived xenograft models. © 2017 Wiley Periodicals, Inc.

  3. Hippocalcin Is Required for Astrocytic Differentiation through Activation of Stat3 in Hippocampal Neural Precursor Cells

    PubMed Central

    Kang, Min-Jeong; Park, Shin-Young; Han, Joong-Soo

    2016-01-01

    Hippocalcin (Hpca) is a neuronal calcium sensor protein expressed in the mammalian brain. However, its function in neural stem/precursor cells has not yet been studied. Here, we clarify the function of Hpca in astrocytic differentiation in hippocampal neural precursor cells (HNPCs). When we overexpressed Hpca in HNPCs in the presence or absence of bFGF, expression levels of nerve-growth factors such as neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5), and brain-derived neurotrophic factor (BDNF), together with the proneural basic helix loop helix (bHLH) transcription factors NeuroD and neurogenin 1 (Ngn1), increased significantly. In addition, there was an increase in the number of cells expressing glial fibrillary acidic protein (GFAP), an astrocyte marker, and in branch outgrowth, indicating astrocytic differentiation of the HNPCs. Downregulation of Hpca by transfection with Hpca siRNA reduced expression of NT-3, NT-4/5, BDNF, NeuroD, and Ngn1 as well as levels of GFAP protein. Furthermore, overexpression of Hpca increased the phosphorylation of STAT3 (Ser727), and this effect was abolished by treatment with a STAT3 inhibitor (S3I-201), suggesting that STAT3 (Ser727) activation is involved in Hpca-mediated astrocytic differentiation. As expected, treatment with Stat3 siRNA or STAT3 inhibitor caused a complete inhibition of astrogliogenesis induced by Hpca overexpression. Taken together, this is the first report to show that Hpca, acting through Stat3, has an important role in the expression of neurotrophins and proneural bHLH transcription factors, and that it is an essential regulator of astrocytic differentiation and branch outgrowth in HNPCs. PMID:27840601

  4. Structural characterization of the human platelet-derived growth factor A-chain cDNA and gene: Alternative exon usage predicts two different precursor proteins

    SciTech Connect

    Rorsman, F.; Bywater, M.; Knott, T.J.; Scott, J.; Betsholtz, C.

    1988-02-01

    The human platelet-derived growth factor (PDGF) A-chain locus was characterized by restriction endonuclease analysis, and the nucleotide sequence of its exons was determined. Seven exons were identified, spanning approximately 22 kilobase pairs of genomic DNA. Alternative exon usage, identified by cDNA cloning, occurs in a human glioblastoma cell line and may give rise to two types of A-chain precursors with different C termini. The exon-intron arrangement was similar to that of the PDGF B-chain/sis locus and seemed to divide the precursor proteins into functional domains. Southern blot analysis of genomic DNA showed that a single PDGF A-chain gene was present in the human genome.

  5. Delayed Transplantation of Human Neural Precursor Cells Improves Outcome from Focal Cerebral Ischemia in Aged Rats

    PubMed Central

    Jin, Kunlin; Mao, XiaoOu; Xie, Lin; Greenberg, Rose B.; Peng, Botao; Moore, Alexander; Greenberg, Maeve B.; Greenberg, David A.

    2010-01-01

    SUMMARY Neural precursor cell (NPC) transplantation may have a role in restoring brain function after stroke, but how aging might affect the brain’s receptivity to such transplants is unknown. We reported previously that transplantation of human embryonic stem cell (hESC)-derived NPCs together with biomaterial (Matrigel) scaffolding into the brains of young adult Sprague-Dawley rats 3 wks after distal middle cerebral artery occlusion (MCAO) reduced infarct volume, and improved neurobehavioral performance. In this study we compared the effect of NPC and Matrigel transplants in young adult (3-mo-old) and aged (24-mo-old) Fisher 344 rats from the National Institute on Aging’s aged rodent colony. Distal MCAO was induced by electrocoagulation and hESC-derived NPCs were transplanted into the infarct cavity 3 wks later. Aged rats developed larger infarcts, but infarct volume and performance on the cylinder and elevated body swing tests, measured 6–8 wks post-transplant, were improved by transplantation. We conclude that advanced age does not preclude a beneficial response to NPC and Matrigel transplantation following experimental stroke. PMID:20883527

  6. cGMP production of patient-specific iPSCs and photoreceptor precursor cells to treat retinal degenerative blindness

    PubMed Central

    Wiley, Luke A.; Burnight, Erin R.; DeLuca, Adam P.; Anfinson, Kristin R.; Cranston, Cathryn M.; Kaalberg, Emily E.; Penticoff, Jessica A.; Affatigato, Louisa M.; Mullins, Robert F.; Stone, Edwin M.; Tucker, Budd A.

    2016-01-01

    Immunologically-matched, induced pluripotent stem cell (iPSC)-derived photoreceptor precursor cells have the potential to restore vision to patients with retinal degenerative diseases like retinitis pigmentosa. The purpose of this study was to develop clinically-compatible methods for manufacturing photoreceptor precursor cells from adult skin in a non-profit cGMP environment. Biopsies were obtained from 35 adult patients with inherited retinal degeneration and fibroblast lines were established under ISO class 5 cGMP conditions. Patient-specific iPSCs were then generated, clonally expanded and validated. Post-mitotic photoreceptor precursor cells were generated using a stepwise cGMP-compliant 3D differentiation protocol. The recapitulation of the enhanced S-cone phenotype in retinal organoids generated from a patient with NR2E3 mutations demonstrated the fidelity of these protocols. Transplantation into immune compromised animals revealed no evidence of abnormal proliferation or tumor formation. These studies will enable clinical trials to test the safety and efficiency of patient-specific photoreceptor cell replacement in humans. PMID:27471043

  7. cGMP production of patient-specific iPSCs and photoreceptor precursor cells to treat retinal degenerative blindness.

    PubMed

    Wiley, Luke A; Burnight, Erin R; DeLuca, Adam P; Anfinson, Kristin R; Cranston, Cathryn M; Kaalberg, Emily E; Penticoff, Jessica A; Affatigato, Louisa M; Mullins, Robert F; Stone, Edwin M; Tucker, Budd A

    2016-07-29

    Immunologically-matched, induced pluripotent stem cell (iPSC)-derived photoreceptor precursor cells have the potential to restore vision to patients with retinal degenerative diseases like retinitis pigmentosa. The purpose of this study was to develop clinically-compatible methods for manufacturing photoreceptor precursor cells from adult skin in a non-profit cGMP environment. Biopsies were obtained from 35 adult patients with inherited retinal degeneration and fibroblast lines were established under ISO class 5 cGMP conditions. Patient-specific iPSCs were then generated, clonally expanded and validated. Post-mitotic photoreceptor precursor cells were generated using a stepwise cGMP-compliant 3D differentiation protocol. The recapitulation of the enhanced S-cone phenotype in retinal organoids generated from a patient with NR2E3 mutations demonstrated the fidelity of these protocols. Transplantation into immune compromised animals revealed no evidence of abnormal proliferation or tumor formation. These studies will enable clinical trials to test the safety and efficiency of patient-specific photoreceptor cell replacement in humans.

  8. Rejuvenation of MPTP-induced human neural precursor cell senescence by activating autophagy.

    PubMed

    Zhu, Liang; Dong, Chuanming; Sun, Chenxi; Ma, Rongjie; Yang, Danjing; Zhu, Hongwen; Xu, Jun

    2015-08-21

    Aging of neural stem cell, which can affect brain homeostasis, may be caused by many cellular mechanisms. Autophagy dysfunction was found in aged and neurodegenerative brains. However, little is known about the relationship between autophagy and human neural stem cell (hNSC) aging. The present study used 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) to treat neural precursor cells (NPCs) derived from human embryonic stem cell (hESC) line H9 and investigate related molecular mechanisms involved in this process. MPTP-treated NPCs were found to undergo premature senescence [determined by increased senescence-associated-β-galactosidase (SA-β-gal) activity, elevated intracellular reactive oxygen species level, and decreased proliferation] and were associated with impaired autophagy. Additionally, the cellular senescence phenotypes were manifested at the molecular level by a significant increase in p21 and p53 expression, a decrease in SOD2 expression, and a decrease in expression of some key autophagy-related genes such as Atg5, Atg7, Atg12, and Beclin 1. Furthermore, we found that the senescence-like phenotype of MPTP-treated hNPCs was rejuvenated through treatment with a well-known autophagy enhancer rapamycin, which was blocked by suppression of essential autophagy gene Beclin 1. Taken together, these findings reveal the critical role of autophagy in the process of hNSC aging, and this process can be reversed by activating autophagy. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Grafting neural precursor cells promotes functional recovery in an SCA1 mouse model.

    PubMed

    Chintawar, Satyan; Hourez, Raphael; Ravella, Ajay; Gall, David; Orduz, David; Rai, Myriam; Bishop, Don Patrick; Geuna, Stefano; Schiffmann, Serge N; Pandolfo, Massimo

    2009-10-21

    The B05 transgenic SCA1 mice, expressing human ataxin-1 with an expanded polyglutamine tract in cerebellar Purkinje cells (PCs), recapitulate many pathological and behavioral characteristics of the neurodegenerative disease spinocerebellar ataxia type 1 (SCA1), including progressive ataxia and PC loss. We transplanted neural precursor cells (NPCs) derived from the subventricular zone of GFP-expressing adult mice into the cerebellar white matter of SCA1 mice when they showed absent (5 weeks), initial (13 weeks), and significant (24 weeks) PC loss. Only in mice with significant cell loss, grafted NPCs migrated into the cerebellar cortex. These animals showed improved motor skills compared with sham-treated controls. No grafted cell adopted the morphological and immunohistochemical characteristics of PCs, but the cerebellar cortex in NPC-grafted SCA1 mice had a significantly thicker molecular layer and more surviving PCs. Perforated patch-clamp recordings revealed a normalization of the PC basal membrane potential, which was abnormally depolarized in sham-treated animals. No significant increase in levels of several neurotrophic factors was observed, suggesting, along with morphological observation, that the neuroprotective effect of grafted NPCs was mediated by direct contact with the host PCs. We postulate that a similar neuroprotective effect of NPCs may be applicable to other cerebellar degenerative diseases.

  10. Smooth Muscle Cells Give Rise to Osteochondrogenic Precursors and Chondrocytes in Calcifying Arteries

    PubMed Central

    Speer, Mei Y.; Yang, Hsueh-Ying; Brabb, Thea; Leaf, Elizabeth; Look, Amy; Lin, Wei-Ling; Frutkin, Andrew; Dichek, David; Giachelli, Cecilia M.

    2009-01-01

    Vascular calcification is a major risk factor for cardiovascular morbidity and mortality. In order to develop appropriate prevention and/or therapeutic strategies for vascular calcification, it is important to understand the origins of the cells that participate in this process. In this report, we used the SM22-Cre recombinase and Rosa26-LacZ alleles to genetically trace cells derived from smooth muscle. We found that smooth muscle cells (SMCs) gave rise to osteochondrogenic precursor- and chondrocyte-like cells in calcified blood vessels of matrix Gla protein deficient (MGP−/−) mice. This lineage reprogramming of SMCs occurred prior to calcium deposition, and was associated with an early onset of Runx2/Cbfa1 expression and the down regulation of myocardin and Msx2. There was no change in the constitutive expression of Sox9 or BMP2. Osterix, Wnt3a and Wnt7a mRNAs were not detected in either calcified MGP−/− or non-calcified wild type (MGP+/+) vessels. Finally, mechanistic studies in vitro suggest that Erk signaling might be required for SMC transdifferentiation under calcifying conditions. These results provide strong support for the hypothesis that adult SMCs can transdifferentiate and that SMC transdifferentiation is an important process driving vascular calcification and the appearance of skeletal elements in calcified vascular lesions. PMID:19197075

  11. Adult-derived stem cells and their potential for use in tissue repair and molecular medicine.

    PubMed

    Young, Henry E; Duplaa, Cecile; Katz, Ryan; Thompson, Tina; Hawkins, Kristina C; Boev, Angel N; Henson, Nicholas L; Heaton, Matthew; Sood, Rajiv; Ashley, Dennis; Stout, Christopher; Morgan, Joe H; Uchakin, Peter N; Rimando, Marylen; Long, Gypsy F; Thomas, Crystal; Yoon, Jee-In; Park, Ji Eun; Hunt, Darren J; Walsh, Nancy M; Davis, Josh C; Lightner, Joel E; Hutchings, Anna M; Murphy, Meredith L; Boswell, Elizabeth; McAbee, Jessica A; Gray, Brandon M; Piskurich, Janet; Blake, Lisa; Collins, Julie A; Moreau, Catherine; Hixson, Douglas; Bowyer, Frank P; Black, Asa C

    2005-01-01

    This report reviews three categories of precursor cells present within adults. The first category of precursor cell, the epiblast-like stem cell, has the potential of forming cells from all three embryonic germ layer lineages, e.g., ectoderm, mesoderm, and endoderm. The second category of precursor cell, the germ layer lineage stem cell, consists of three separate cells. Each of the three cells is committed to form cells limited to a specific embryonic germ layer lineage. Thus the second category consists of germ layer lineage ectodermal stem cells, germ layer lineage mesodermal stem cells, and germ layer lineage endodermal stem cells. The third category of precursor cells, progenitor cells, contains a multitude of cells. These cells are committed to form specific cell and tissue types and are the immediate precursors to the differentiated cells and tissues of the adult. The three categories of precursor cells can be readily isolated from adult tissues. They can be distinguished from each other based on their size, growth in cell culture, expressed genes, cell surface markers, and potential for differentiation. This report also discusses new findings. These findings include the karyotypic analysis of germ layer lineage stem cells; the appearance of dopaminergic neurons after implantation of naive adult pluripotent stem cells into a 6-hydroxydopamine-lesioned Parkinson's model; and the use of adult stem cells as transport mechanisms for exogenous genetic material. We conclude by discussing the potential roles of adult-derived precursor cells as building blocks for tissue repair and as delivery vehicles for molecular medicine.

  12. Early B-lymphocyte precursor cells in mouse bone marrow: Subosteal localization of B220+ cells during postirradiation regeneration

    SciTech Connect

    Jacobsen, K.; Tepper, J.; Osmond, D.G. )

    1990-05-01

    The localization of early B-lymphocyte precursor cells in the bone marrow of young mice has been studied during recovery from sublethal whole body gamma-irradiation (150 rad). Initial studies by double immunofluorescence labeling of the B-lineage-associated cell surface glycoprotein, B220, and of mu heavy chains in bone marrow cell suspensions, demonstrated a sequential wave of regeneration of early B precursor cells, pre-B cells, and B cells. Early B precursor cells expressing B220 but not mu chains were enriched at 1-3 days following irradiation. After in vivo administration of 125I-labeled monoclonal antibody 14.8 to detect B220+ cells in situ, light and electron microscope radioautography of femoral bone marrow sections revealed concentrations of labeled B220+ cells located peripherally near the cortical bone at 1-3 days following irradiation, increasing in numbers in more central areas by 5-7 days. Proliferative B220+ precursor cells were found within layers of bone-lining cells and in a subosteal area characterized by a prominent electron-dense extracellular matrix, often associated with stromal reticular cells. The results demonstrate that the precursor cells that are active in the bone marrow early in the recovery of B lymphopoiesis after gamma-irradiation are located both within and near the endosteum of the surrounding bone. The distinctive extracellular matrix and stromal cell associations noted in this region may contribute to a supportive local microenvironment for early hemopoietic progenitor cells.

  13. Sparing of extraocular muscle in aging and muscular dystrophies: a myogenic precursor cell hypothesis.

    PubMed

    Kallestad, Kristen M; Hebert, Sadie L; McDonald, Abby A; Daniel, Mark L; Cu, Sharon R; McLoon, Linda K

    2011-04-01

    The extraocular muscles (EOM) are spared from pathology in aging and many forms of muscular dystrophy. Despite many studies, this sparing remains an enigma. The EOM have a distinct embryonic lineage compared to somite-derived muscles, and we have shown that they continuously remodel throughout life, maintaining a population of activated satellite cells even in aging. These data suggested the hypothesis that there is a population of myogenic precursor cells (mpcs) in EOM that is different from those in limb, with either elevated numbers of stem cells and/or mpcs with superior proliferative capacity compared to mpcs in limb. Using flow cytometry, EOM and limb muscle mononuclear cells were compared, and a number of differences were seen. Using two different cell isolation methods, EOM have significantly more mpcs per mg muscle than limb skeletal muscle. One specific subpopulation significantly increased in EOM compared to limb was positive for CD34 and negative for Sca-1, M-cadherin, CD31, and CD45. We named these the EOMCD34 cells. Similar percentages of EOMCD34 cells were present in both newborn EOM and limb muscle. They were retained in aged EOM, whereas the population decreased significantly in adult limb muscle and were extremely scarce in aged limb muscle. Most importantly, the percentage of EOMCD34 cells was elevated in the EOM from both the mdx and the mdx/utrophin(-/-) (DKO) mouse models of DMD and extremely scarce in the limb muscles of these mice. In vitro, the EOMCD34 cells had myogenic potential, forming myotubes in differentiation media. After determining a media better able to induce proliferation in these cells, a fusion index was calculated. The cells isolated from EOM had a 40% higher fusion index compared to the same cells isolated from limb muscle. The EOMCD34 cells were resistant to both oxidative stress and mechanical injury. These data support our hypothesis that the EOM may be spared in aging and in muscular dystrophies due to a subpopulation

  14. Sparing of extraocular muscle in aging and muscular dystrophies: A myogenic precursor cell hypothesis

    SciTech Connect

    Kallestad, Kristen M.; Hebert, Sadie L.; McDonald, Abby A.; Daniel, Mark L.; Cu, Sharon R.; McLoon, Linda K.

    2011-04-01

    The extraocular muscles (EOM) are spared from pathology in aging and many forms of muscular dystrophy. Despite many studies, this sparing remains an enigma. The EOM have a distinct embryonic lineage compared to somite-derived muscles, and we have shown that they continuously remodel throughout life, maintaining a population of activated satellite cells even in aging. These data suggested the hypothesis that there is a population of myogenic precursor cells (mpcs) in EOM that is different from those in limb, with either elevated numbers of stem cells and/or mpcs with superior proliferative capacity compared to mpcs in limb. Using flow cytometry, EOM and limb muscle mononuclear cells were compared, and a number of differences were seen. Using two different cell isolation methods, EOM have significantly more mpcs per mg muscle than limb skeletal muscle. One specific subpopulation significantly increased in EOM compared to limb was positive for CD34 and negative for Sca-1, M-cadherin, CD31, and CD45. We named these the EOMCD34 cells. Similar percentages of EOMCD34 cells were present in both newborn EOM and limb muscle. They were retained in aged EOM, whereas the population decreased significantly in adult limb muscle and were extremely scarce in aged limb muscle. Most importantly, the percentage of EOMCD34 cells was elevated in the EOM from both the mdx and the mdx/utrophin{sup -/-} (DKO) mouse models of DMD and extremely scarce in the limb muscles of these mice. In vitro, the EOMCD34 cells had myogenic potential, forming myotubes in differentiation media. After determining a media better able to induce proliferation in these cells, a fusion index was calculated. The cells isolated from EOM had a 40% higher fusion index compared to the same cells isolated from limb muscle. The EOMCD34 cells were resistant to both oxidative stress and mechanical injury. These data support our hypothesis that the EOM may be spared in aging and in muscular dystrophies due to a

  15. Dual Targeting of Cell Wall Precursors by Teixobactin Leads to Cell Lysis

    PubMed Central

    Homma, Tomoyuki; Nuxoll, Austin; Gandt, Autumn Brown; Ebner, Patrick; Engels, Ina; Schneider, Tanja; Götz, Friedrich; Lewis, Kim

    2016-01-01

    Teixobactin represents the first member of a newly discovered class of antibiotics that act through inhibition of cell wall synthesis. Teixobactin binds multiple bactoprenol-coupled cell wall precursors, inhibiting both peptidoglycan and teichoic acid synthesis. Here, we show that the impressive bactericidal activity of teixobactin is due to the synergistic inhibition of both targets, resulting in cell wall damage, delocalization of autolysins, and subsequent cell lysis. We also find that teixobactin does not bind mature peptidoglycan, further increasing its activity at high cell densities and against vancomycin-intermediate Staphylococcus aureus (VISA) isolates with thickened peptidoglycan layers. These findings add to the attractiveness of teixobactin as a potential therapeutic agent for the treatment of infection caused by antibiotic-resistant Gram-positive pathogens. PMID:27550357

  16. Precursor Derived Nanostructured Si-C-X Materials for Nuclear Applications. Final Report, October 2010 - September 2014

    SciTech Connect

    Bordia, Rajendra; Tomar, Vikas; Henager, Chuck

    2015-04-08

    Polymer derived ceramic route is an attractive approach to make structural materials with unique nanostructures that have very desirable high temperature properties. Processing techniques to make a variety of needed shapes and forms (e.g. coatings, matrices for fiber reinforced composites, porous ceramics) have been developed. With appropriate high temperature processing, the precursors can be converted to nano-crystalline materials. In this collaborative project, we investigated the processing, stability and properties of nanostructured Si-C materials, derived from polymeric precursors, and their performance under conditions appropriate for nuclear energy applications. All the milestones of the project were accomplished. Some of the results are being currently analyzed and additional papers being prepared in which support from NEUP will be acknowledged. So far, eight peer-reviewed papers have been published and one invention disclosure made. In this report, we summarize the major findings of this project.

  17. Differentiation of Donor-Derived Cells Into Microglia After Umbilical Cord Blood Stem Cell Transplantation

    PubMed Central

    Takahashi, Kazuya; Kakuda, Yumiko; Munemoto, Saori; Yamazaki, Hirohito; Nozaki, Ichiro; Yamada, Masahito

    2015-01-01

    Abstract Recent studies have indicated that microglia originate from immature progenitors in the yolk sac. After birth, microglial populations are maintained under normal conditions via self-renewal without the need to recruit monocyte-derived microglial precursors. Peripheral cell invasion of the brain parenchyma can only occur with disruption of the blood-brain barrier. Here, we report an autopsy case of an umbilical cord blood transplant recipient in whom cells derived from the donor blood differentiated into ramified microglia in the recipient brain parenchyma. Although the blood-brain barrier and glia limitans seemed to prevent invasion of these donor-derived cells, most of the invading donor-derived ramified cells were maintained in the cerebral cortex. This result suggests that invasion of donor-derived cells occurs through the pial membrane. PMID:26226134

  18. Regulation of Asymmetric Cell Division in Mammalian Neural Stem and Cancer Precursor Cells.

    PubMed

    Daynac, Mathieu; Petritsch, Claudia K

    Stem and progenitor cells are characterized by their abilities to self-renew and produce differentiated progeny. The balance between self-renewal and differentiation is achieved through control of cell division mode, which can be either asymmetric or symmetric. Failure to properly control cell division mode may result in premature depletion of the stem/progenitor cell pool or abnormal growth and impaired differentiation. In many tissues, including the brain, stem cells and progenitor cells undergo asymmetric cell division through the establishment of cell polarity. Cell polarity proteins are therefore potentially critical regulators of asymmetric cell division. Decrease or loss of asymmetric cell division can be associated with reduced differentiation common during aging or impaired remyelination as seen in demyelinating diseases. Progenitor-like glioma precursor cells show decreased asymmetric cell division rates and increased symmetric divisions, which suggests that asymmetric cell division suppresses brain tumor formation. Cancer stem cells, on the other hand, still undergo low rates of asymmetric cell division, which may provide them with a survival advantage during therapy. These findings led to the hypotheses that asymmetric cell divisions are not always tumor suppressive but can also be utilized to maintain a cancer stem cell population. Proper control of cell division mode is therefore not only deemed necessary to generate cellular diversity during development and to maintain adult tissue homeostasis but may also prevent disease and determine disease progression. Since brain cancer is most common in the adult and aging population, we review here the current knowledge on molecular mechanisms that regulate asymmetric cell divisions in the neural and oligodendroglial lineage during development and in the adult brain.

  19. PDGFRα(+) Cells in Embryonic Stem Cell Cultures Represent the In Vitro Equivalent of the Pre-implantation Primitive Endoderm Precursors.

    PubMed

    Lo Nigro, Antonio; de Jaime-Soguero, Anchel; Khoueiry, Rita; Cho, Dong Seong; Ferlazzo, Giorgia Maria; Perini, Ilaria; Abon Escalona, Vanesa; Aranguren, Xabier Lopez; Chuva de Sousa Lopes, Susana M; Koh, Kian Peng; Conaldi, Pier Giulio; Hu, Wei-Shou; Zwijsen, An; Lluis, Frederic; Verfaillie, Catherine M

    2017-02-14

    In early mouse pre-implantation development, primitive endoderm (PrE) precursors are platelet-derived growth factor receptor alpha (PDGFRα) positive. Here, we demonstrated that cultured mouse embryonic stem cells (mESCs) express PDGFRα heterogeneously, fluctuating between a PDGFRα+ (PrE-primed) and a platelet endothelial cell adhesion molecule 1 (PECAM1)-positive state (epiblast-primed). The two surface markers can be co-detected on a third subpopulation, expressing epiblast and PrE determinants (double-positive). In vitro, these subpopulations differ in their self-renewal and differentiation capability, transcriptional and epigenetic states. In vivo, double-positive cells contributed to epiblast and PrE, while PrE-primed cells exclusively contributed to PrE derivatives. The transcriptome of PDGFRα(+) subpopulations differs from previously described subpopulations and shows similarities with early/mid blastocyst cells. The heterogeneity did not depend on PDGFRα but on leukemia inhibitory factor and fibroblast growth factor signaling and DNA methylation. Thus, PDGFRα(+) cells represent the in vitro counterpart of in vivo PrE precursors, and their selection from cultured mESCs yields pure PrE precursors.

  20. Mesenchymal precursor cells maintain the differentiation and proliferation potentials of breast epithelial cells

    PubMed Central

    2014-01-01

    Introduction Stromal-epithelial interactions play a fundamental role in tissue homeostasis, controlling cell proliferation and differentiation. Not surprisingly, aberrant stromal-epithelial interactions contribute to malignancies. Studies of the cellular and molecular mechanisms underlying these interactions require ex vivo experimental model systems that recapitulate the complexity of human tissue without compromising the differentiation and proliferation potentials of human primary cells. Methods We isolated and characterized human breast epithelial and mesenchymal precursors from reduction mammoplasty tissue and tagged them with lentiviral vectors. We assembled heterotypic co-cultures and compared mesenchymal and epithelial cells to cells in corresponding monocultures by analyzing growth, differentiation potentials, and gene expression profiles. Results We show that heterotypic culture of non-immortalized human primary breast epithelial and mesenchymal precursors maintains their proliferation and differentiation potentials and constrains their growth. We further describe the gene expression profiles of stromal and epithelial cells in co-cultures and monocultures and show increased expression of the tumor growth factor beta (TGFβ) family member inhibin beta A (INHBA) in mesenchymal cells grown as co-cultures compared with monocultures. Notably, overexpression of INHBA in mesenchymal cells increases colony formation potential of epithelial cells, suggesting that it contributes to the dynamic reciprocity between breast mesenchymal and epithelial cells. Conclusions The described heterotypic co-culture system will prove useful for further characterization of the molecular mechanisms mediating interactions between human normal or neoplastic breast epithelial cells and the stroma, and will provide a framework to test the relevance of the ever-increasing number of oncogenomic alterations identified in human breast cancer. PMID:24916766

  1. Mesenchymal precursor cells maintain the differentiation and proliferation potentials of breast epithelial cells.

    PubMed

    Duss, Stephan; Brinkhaus, Heike; Britschgi, Adrian; Cabuy, Erik; Frey, Daniel M; Schaefer, Dirk J; Bentires-Alj, Mohamed

    2014-06-10

    Stromal-epithelial interactions play a fundamental role in tissue homeostasis, controlling cell proliferation and differentiation. Not surprisingly, aberrant stromal-epithelial interactions contribute to malignancies. Studies of the cellular and molecular mechanisms underlying these interactions require ex vivo experimental model systems that recapitulate the complexity of human tissue without compromising the differentiation and proliferation potentials of human primary cells. We isolated and characterized human breast epithelial and mesenchymal precursors from reduction mammoplasty tissue and tagged them with lentiviral vectors. We assembled heterotypic co-cultures and compared mesenchymal and epithelial cells to cells in corresponding monocultures by analyzing growth, differentiation potentials, and gene expression profiles. We show that heterotypic culture of non-immortalized human primary breast epithelial and mesenchymal precursors maintains their proliferation and differentiation potentials and constrains their growth. We further describe the gene expression profiles of stromal and epithelial cells in co-cultures and monocultures and show increased expression of the tumor growth factor beta (TGFβ) family member inhibin beta A (INHBA) in mesenchymal cells grown as co-cultures compared with monocultures. Notably, overexpression of INHBA in mesenchymal cells increases colony formation potential of epithelial cells, suggesting that it contributes to the dynamic reciprocity between breast mesenchymal and epithelial cells. The described heterotypic co-culture system will prove useful for further characterization of the molecular mechanisms mediating interactions between human normal or neoplastic breast epithelial cells and the stroma, and will provide a framework to test the relevance of the ever-increasing number of oncogenomic alterations identified in human breast cancer.

  2. Telencephalic neural precursor cells show transient competence to interpret the dopaminergic niche of the embryonic midbrain.

    PubMed

    Baizabal, José-Manuel; Valencia, Concepción; Guerrero-Flores, Gilda; Covarrubias, Luis

    2011-01-15

    Neural Precursor Cells (NPCs) generate complex stereotypic arrays of neuronal subtypes in the brain. This process involves the integration of patterning cues that progressively restrict the fate of specific NPCs. Yet the capacity of NPCs to interpret foreign microenvironments during development remains poorly defined. The aim of this work was to test the competence of mouse telencephalic NPCs to respond to the dopaminergic niche of the mesencephalon. Telencephalic NPCs isolated from midgestation mouse embryos (E10.5) and transplanted to age-matched mesencephalic explants efficiently differentiated into neurons but were largely unable to produce midbrain dopaminergic (mDA) neurons. Instead, E10.5 telencephalic NPCs behaved as restricted gabaergic progenitors that maintained ectopic expression of Foxg1 and Pax6. In contrast, E8.5 telencephalic NPCs were able to differentiate into Lmx1a(+)/Foxa2(+)/TH(+) neurons in the dopaminergic niche of the mesencephalic explants. In addition, these early telencephalic NPCs showed region-dependent expression of Nkx6.1, Nkx2.2 and site-specific differentiation into gabaergic neurons within the mesencephalic tissue. Significant dopaminergic differentiation of E8.5 telencephalic NPCs was not observed after transplantation to E12.5 mesencephalic explants, suggesting that inductive signals in the dopaminergic niche rapidly decay after midgestation. Moreover, we employed transplantation of embryonic stem cells-derived precursors to demonstrate that extinction of inductive signals within the telencephalon lags behind the commitment of residing NPCs. Our data indicate that the plasticity to interpret multiple instructive niches is an early and ephemeral feature of the telencephalic neural lineage.

  3. Antioxidant Protection of NADPH-Depleted Oligodendrocyte Precursor Cells Is Dependent on Supply of Reduced Glutathione

    PubMed Central

    Kilanczyk, Ewa; Saraswat Ohri, Sujata; Whittemore, Scott R.

    2016-01-01

    The pentose phosphate pathway is the main source of NADPH, which by reducing oxidized glutathione, contributes to antioxidant defenses. Although oxidative stress plays a major role in white matter injury, significance of NADPH for oligodendrocyte survival has not been yet investigated. It is reported here that the NADPH antimetabolite 6-amino-NADP (6AN) was cytotoxic to cultured adult rat spinal cord oligodendrocyte precursor cells (OPCs) as well as OPC-derived oligodendrocytes. The 6AN-induced necrosis was preceded by increased production of superoxide, NADPH depletion, and lower supply of reduced glutathione. Moreover, survival of NADPH-depleted OPCs was improved by the antioxidant drug trolox. Such cells were also protected by physiological concentrations of the neurosteroid dehydroepiandrosterone (10−8 M). The protection by dehydroepiandrosterone was associated with restoration of reduced glutathione, but not NADPH, and was sensitive to inhibition of glutathione synthesis. A similar protective mechanism was engaged by the cAMP activator forskolin or the G protein-coupled estrogen receptor (GPER/GPR30) ligand G1. Finally, treatment with the glutathione precursor N-acetyl cysteine reduced cytotoxicity of 6AN. Taken together, NADPH is critical for survival of OPCs by supporting their antioxidant defenses. Consequently, injury-associated inhibition of the pentose phosphate pathway may be detrimental for the myelination or remyelination potential of the white matter. Conversely, steroid hormones and cAMP activators may promote survival of NADPH-deprived OPCs by increasing a NADPH-independent supply of reduced glutathione. Therefore, maintenance of glutathione homeostasis appears as a critical effector mechanism for OPC protection against NADPH depletion and preservation of the regenerative potential of the injured white matter. PMID:27449129

  4. New insights into glycopeptide antibiotic binding to cell wall precursors using SPR and NMR spectroscopy.

    PubMed

    Treviño, Juan; Bayón, Carlos; Ardá, Ana; Marinelli, Flavia; Gandolfi, Raffaella; Molinari, Francesco; Jimenez-Barbero, Jesús; Hernáiz, María J

    2014-06-10

    Glycopeptide antibiotics, such as vancomycin and teicoplanin, are used to treat life-threatening infections caused by multidrug-resistant Gram-positive pathogens. They inhibit bacterial cell wall biosynthesis by binding to the D-Ala-D-Ala C-terminus of peptidoglycan precursors. Vancomycin-resistant bacteria replace the dipeptide with the D-Ala-D-Lac depsipeptide, thus reducing the binding affinity of the antibiotics with their molecular targets. Herein, studies of the interaction of teicoplanin, teicoplanin-like A40926, and of their semisynthetic derivatives (mideplanin, MDL63,246, dalbavancin) with peptide analogues of cell-wall precursors by NMR spectroscopy and surface plasmon resonance (SPR) are reported. NMR spectroscopy revealed the existence of two different complexes in solution, when the different glycopeptides interact with Ac2KdAlaDAlaOH. Despite the NMR experimental conditions, which are different from those employed for the SPR measurements, the NMR spectroscopy results parallel those deduced in the chip with respect to the drastic binding difference existing between the D-Ala and the D-Lac terminating analogues, confirming that all these antibiotics share the same primary molecular mechanism of action and resistance. Kinetic analysis of the interaction between the glycopeptide antibiotics and immobilized AcKdAlaDAlaOH by SPR suggest a dimerization process that was not observed by NMR spectroscopy in DMSO solution. Moreover, in SPR, all glycopeptides with a hydrophobic acyl chain present stronger binding with a hydrophobic surface than vancomycin, indicating that additional interactions through the employed surface are involved. In conclusion, SPR provides a tool to differentiate between vancomycin and other glycopeptides, and the calculated binding affinities at the surface seem to be more relevant to in vitro antimicrobial activity than the estimations from NMR spectroscopy analysis.

  5. Myeloid-derived suppressor cells

    PubMed Central

    Chandra, Dinesh; Gravekamp, Claudia

    2013-01-01

    While conventional anticancer therapies, including surgical resection, radiotherapy, and/or chemotherapy, are relatively efficient at eliminating primary tumors, these treatment modalities are largely ineffective against metastases. At least in part, this reflects the rather inefficient delivery of conventional anticancer agents to metastatic lesions. We have recently demonstrated that myeloid-derived suppressor cells (MDSCs) can be used as cellular missiles to selectively deliver a radioisotope-coupled attenuated variant of Listeria monocytogenes to both primary and metastatic neoplastic lesions in mice with pancreatic cancer. This novel immunotherapeutic intervention robustly inhibited tumor growth while promoting a dramatic decrease in the number of metastases. PMID:24427545

  6. Co-ultramicronized Palmitoylethanolamide/Luteolin Promotes the Maturation of Oligodendrocyte Precursor Cells.

    PubMed

    Barbierato, Massimo; Facci, Laura; Marinelli, Carla; Zusso, Morena; Argentini, Carla; Skaper, Stephen D; Giusti, Pietro

    2015-11-18

    Oligodendrocytes have limited ability to repair the damage to themselves or to other nerve cells, as seen in demyelinating diseases like multiple sclerosis. An important strategy may be to replace the lost oligodendrocytes and/or promote the maturation of undifferentiated oligodendrocyte precursor cells (OPCs). Recent studies show that a composite of co-ultramicronized N-palmitoylethanolamine (PEA) and luteolin (co-ultramicronized PEA/luteolin, 10:1 by mass) is efficacious in improving outcome in experimental models of spinal cord and traumatic brain injuries. Here, we examined the ability of co-ultramicronized PEA/luteolin to promote progression of OPCs into a more differentiated phenotype. OPCs derived from newborn rat cortex were placed in culture and treated the following day with 10 μM co-ultramicronized PEA/luteolin. Cells were collected 1, 4 and 8 days later and analyzed for expression of myelin basic protein (MBP). qPCR and Western blot analyses revealed a time-dependent increase in expression of both mRNA for MBP and MBP content, along with an increased expression of genes involved in lipid biogenesis. Ultramicronized PEA or luteolin, either singly or in simple combination, were ineffective. Further, co-ultramicronized PEA/luteolin promoted morphological development of OPCs and total protein content without affecting proliferation. Co-ultramicronized PEA/luteolin may represent a novel pharmacological strategy to promote OPC maturation.

  7. Co-ultramicronized Palmitoylethanolamide/Luteolin Promotes the Maturation of Oligodendrocyte Precursor Cells

    PubMed Central

    Barbierato, Massimo; Facci, Laura; Marinelli, Carla; Zusso, Morena; Argentini, Carla; Skaper, Stephen D.; Giusti, Pietro

    2015-01-01

    Oligodendrocytes have limited ability to repair the damage to themselves or to other nerve cells, as seen in demyelinating diseases like multiple sclerosis. An important strategy may be to replace the lost oligodendrocytes and/or promote the maturation of undifferentiated oligodendrocyte precursor cells (OPCs). Recent studies show that a composite of co-ultramicronized N-palmitoylethanolamine (PEA) and luteolin (co-ultramicronized PEA/luteolin, 10:1 by mass) is efficacious in improving outcome in experimental models of spinal cord and traumatic brain injuries. Here, we examined the ability of co-ultramicronized PEA/luteolin to promote progression of OPCs into a more differentiated phenotype. OPCs derived from newborn rat cortex were placed in culture and treated the following day with 10 μM co-ultramicronized PEA/luteolin. Cells were collected 1, 4 and 8 days later and analyzed for expression of myelin basic protein (MBP). qPCR and Western blot analyses revealed a time-dependent increase in expression of both mRNA for MBP and MBP content, along with an increased expression of genes involved in lipid biogenesis. Ultramicronized PEA or luteolin, either singly or in simple combination, were ineffective. Further, co-ultramicronized PEA/luteolin promoted morphological development of OPCs and total protein content without affecting proliferation. Co-ultramicronized PEA/luteolin may represent a novel pharmacological strategy to promote OPC maturation. PMID:26578323

  8. c-jun is differentially expressed in embryonic and adult neural precursor cells.

    PubMed

    Kawashima, Fumiaki; Saito, Kengo; Kurata, Hirofumi; Maegaki, Yoshihiro; Mori, Tetsuji

    2017-01-16

    c-jun, a major component of AP-1 transcription factor, has a wide variety of functions. In the embryonic brain, c-jun mRNA is abundantly expressed in germinal layers around the ventricles. Although the subventricular zone (SVZ) of the adult brain is a derivative of embryonic germinal layers and contains neural precursor cells (NPCs), the c-jun expression pattern is not clear. To study the function of c-jun in adult neurogenesis, we analyzed c-jun expression in the adult SVZ by immunohistochemistry and compared it with that of the embryonic brain. We found that almost all proliferating embryonic NPCs expressed c-jun, but the number of c-jun immunopositive cells among proliferating adult NPCs was about half. In addition, c-jun was hardly expressed in post-mitotic migrating neurons in the embryonic brain, but the majority of c-jun immunopositive cells were tangentially migrating neuroblasts heading toward the olfactory bulb in the adult brain. In addition, status epilepticus is known to enhance the transient proliferation of adult NPCs, but the c-jun expression pattern was not significantly affected. These expression patterns suggest that c-jun has a pivotal role in the proliferation of embryonic NPCs, but it has also other roles in adult neurogenesis.

  9. Accumulation of specific sterol precursors targets a MAP kinase cascade mediating cell-cell recognition and fusion.

    PubMed

    Weichert, Martin; Lichius, Alexander; Priegnitz, Bert-Ewald; Brandt, Ulrike; Gottschalk, Johannes; Nawrath, Thorben; Groenhagen, Ulrike; Read, Nick D; Schulz, Stefan; Fleißner, André

    2016-10-18

    Sterols are vital components of eukaryotic cell membranes. Defects in sterol biosynthesis, which result in the accumulation of precursor molecules, are commonly associated with cellular disorders and disease. However, the effects of these sterol precursors on the metabolism, signaling, and behavior of cells are only poorly understood. In this study, we show that the accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain specifically disrupts cell-cell communication and fusion in the fungus Neurospora crassa Genetically identical germinating spores of this fungus undergo cell-cell fusion, thereby forming a highly interconnected supracellular network during colony initiation. Before fusion, the cells use an unusual signaling mechanism that involves the coordinated and alternating switching between signal sending and receiving states of the two fusion partners. Accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain disrupts this coordinated cell-cell communication and suppresses cell fusion. These specific sterol precursors target a single ERK-like mitogen-activated protein (MAP) kinase (MAK-1)-signaling cascade, whereas a second MAP kinase pathway (MAK-2), which is also involved in cell fusion, is unaffected. These observations indicate that a minor specific change in sterol structure can exert a strong detrimental effect on a key signaling pathway of the cell, resulting in the absence of cell fusion.

  10. Cytotoxicity of CD56-positive lymphocytes against autologous B-cell precursor acute lymphoblastic leukemia cells.

    PubMed

    Fei, F; Lim, M; George, A A; Kirzner, J; Lee, D; Seeger, R; Groffen, J; Abdel-Azim, H; Heisterkamp, N

    2015-04-01

    Precursor B-lineage acute lymphoblastic leukemia (pre-B ALL) affects hematopoietic development and therefore is associated with immune deficiencies that can be further exacerbated by chemotherapy. It is unclear if and when monoclonal antibodies (mAbs) that stimulate antibody-mediated cellular cytotoxicity (ADCC) can be used for treatment because this depends on the presence of functional effector cells. Here, we used flow cytometry to determine that patient samples at diagnosis, post-induction and relapse contain detectable numbers of CD56+ cells. We were able to selectively expand CD56+ immune effector cells from bone marrow and peripheral blood samples at diagnosis and at various stages of treatment by co-culture with artificial antigen-presenting K562 clone 9.mbIL-21 cells. Amplified CD56+CD3- cells had spontaneous and anti-B cell-activating factor receptor mAb-stimulated ADCC activity against allogeneic ALL cells, which could be further enhanced by IL-15. Importantly, matched CD56+ effector cells also killed autologous ALL cells grown out from leukemia samples of the same patient, through both spontaneous as well as antibody-dependent cellular cytotoxicity. Since autologous cell therapy will not be complicated by graft-versus-host disease, our results show that expanded CD56+ cells could be applied for treatment of pre-B ALL without transplantation, or for purging of bone marrow in the setting of autologous bone marrow transplants.

  11. Stem/precursor cell-based CNS therapy: the importance of circumventing immune suppression by transplanting autologous cells.

    PubMed

    Kulbatski, Iris

    2010-09-01

    Stem/precursor cell (SPC) therapy for neurodegeneration and neurotrauma has enormous therapeutic potential, but despite ongoing research efforts the success of clinical trials remains limited. Therapies that utilize immune suppression in combination with SPC transplantation have thus far failed to consider the beneficial role of the immune system in central nervous system (CNS) recovery. Systemic immune suppression may prevent neural repair, and in some cases exacerbate the underlying disorder. Until about a decade ago, immunosuppression for CNS disorders was viewed as a therapeutic target, based on the perception that all immune activity in the CNS was destructive. However, recent studies show that the infiltration of blood-borne immune cells into the CNS following neurotrauma and during chronic neurodegeneration promote CNS protection and regeneration. In the context of SPC therapies, although immune suppression prevents rejection of non-autologous cell grafts, it also prevents the restorative immune response by eliminating the immune mediated guidance cues that are required for SPCs to migrate to the location they are needed, and preventing SPC-mediated immunomodulation. This article argues in favor of transplanting autologous SPCs, particularly bone marrow derived cells. The therapeutic use of autologous SPCs for neural repair circumvents the need for concomitant immune suppression, exploits the immunomodulatory capacity of these cells, and maintains the immune niche that supports neural repair and is required to guide these cells to their appropriate locations. Overall, such an approach accommodates the requirements for translational therapeutics, and provides a standardized platform for reconciling the inherent controversies in the science.

  12. Establishment of a cell line with features of early dendritic cell precursors from fetal mouse skin.

    PubMed

    Girolomoni, G; Lutz, M B; Pastore, S; Assmann, C U; Cavani, A; Ricciardi-Castagnoli, P

    1995-08-01

    During ontogeny, the skin is progressively populated by major histocompatibility complex class II-negative dendritic cell (DC) precursors that then mature into efficient antigen-presenting cells (APC). To characterize these DC progenitors better, we generated myeloid cell lines from fetal mouse skin by infecting cell suspensions with a retroviral vector carrying an envAKR-mycMH2 fusion gene. These cells, represented by the line FSDC, displayed a dendritic morphology and their proliferation in serum-free medium was promoted by granulocyte/macrophage colony-stimulating factor (GM-CSF), but not macrophage-CSF. FSDC expressed strong surface-membrane ATP/ADPase activity, intracellular staining for 2A1 antigen, and a surface phenotype consistent with a myeloid precursor: H-2d,b+, I-Ad,b+, CD54+, CD11b+, CD11c+, 2.4G2+, F4/80+, CD44+, 2F8+, ER-MP 12-, Sca-1+, Sca-2+, NLDC-145-, B7.2+, B7.1-, J11d-, B220-, Thy-1-, and CD3-. FSDC stimulated poorly allogeneic or syngeneic T cells in the primary mixed-leukocyte reaction, and markedly increased this function after treatment with GM-CSF, GM-CSF and interleukin (IL)-4 or interferon-gamma (IFN-gamma); in contrast, stem cell factor, IL-1 alpha and tumor necrosis factor-alpha had no effect. Preculture with IFN-gamma was required for presentation of haptens to primed T cells in vitro. However, FSDC, even after cytokine activation, were less potent APC than adult epidermal Langerhans cells in both of the above assays. Finally, FSDC derivatized with haptens and injected either intravenously or subcutaneously could efficiently induce contact sensitivity responses in naive syngeneic mice. The results indicate that fetal mouse skin is colonized by myeloid precursors possessing a macrophage/immature DC-like surface phenotype and priming capacity in vivo. These cells need further differentiation and activation signals (e.g. cytokines) to express their antigen presenting potential in vitro.

  13. The thymus exports long-lived fully committed T cell precursors that can colonize primary lymphoid organs.

    PubMed

    Lambolez, Florence; Arcangeli, Marie-Laure; Joret, Anne-Marie; Pasqualetto, Valérie; Cordier, Corinne; Di Santo, James P; Rocha, Benedita; Ezine, Sophie

    2006-01-01

    Thymic export of cells is believed to be restricted to mature T cells. Here we show that the thymus also exports fully committed T cell precursors that colonize primary lymphoid organs. These precursor cells exited the thymus before T cell receptor rearrangements and colonized lymphoid organs such as the thymus and the gut. Migration of the thymic T cell-committed precursors led to permanent colonization of the gut precursor compartment, improved the capacity of gut precursors to further differentiate into T cells and was sufficient for the generation of 'euthymic like' CD8alphaalpha(+) intraepithelial lymphocytes. These data demonstrate a new function for the thymus in peripheral seeding with T cell precursors that become long lived after thymus export.

  14. Cytotoxicity of CD56-positive lymphocytes against autologous B-cell precursor acute lymphoblastic leukemia cells

    PubMed Central

    Fei, Fei; Lim, Min; George, Aswathi A.; Kirzner, Jonathan; Lee, Dean; Seeger, Robert; Groffen, John; Abdel-Azim, Hisham; Heisterkamp, Nora

    2014-01-01

    Precursor B-lineage acute lymphoblastic leukemia (pre-B ALL) affects hematopoietic development and therefore is associated with immune deficiencies that can be further exacerbated by chemotherapy. It is unclear if and when monoclonal antibodies (mAbs) that stimulate antibody-mediated cellular cytotoxicity (ADCC) can be used for treatment because this depends on the presence of functional effector cells. Here, we used flow cytometry to determine that patient samples at diagnosis, post-induction and relapse contain detectable numbers of CD56+ cells. We were able to selectively expand CD56+ immune effector cells from bone marrow and peripheral blood samples at diagnosis and at various stages of treatment by co-culture with artificial antigen-presenting K562 clone 9.mbIL-21 cells. Amplified CD56+CD3- cells had spontaneous and anti-BAFF-R mAb-stimulated ADCC activity against autologous ALL cells, which could be further enhanced by IL15. Importantly, matched CD56+ effector cells also killed autologous ALL cells grown out from leukemia samples of the same patient, through both spontaneous as well as antibody-dependent cellular cytotoxicity. Since autologous cell therapy will not be complicated by graft-versus-host disease, our results show that expanded CD56+ cells could be applied for treatment of pre-B-ALL without transplantation, or for purging of bone marrow in the setting of autologous bone marrow transplants. PMID:25134458

  15. IPC: professional type 1 interferon-producing cells and plasmacytoid dendritic cell precursors.

    PubMed

    Liu, Yong-Jun

    2005-01-01

    Type 1 interferon-(alpha, beta, omega)-producing cells (IPCs), also known as plasmacytoid dendritic cell precursors (pDCs), represent 0.2%-0.8% of peripheral blood mononuclear cells in both humans and mice. IPCs display plasma cell morphology, selectively express Toll-like receptor (TLR)-7 and TLR9, and are specialized in rapidly secreting massive amounts of type 1 interferon following viral stimulation. IPCs can promote the function of natural killer cells, B cells, T cells, and myeloid DCs through type 1 interferons during an antiviral immune response. At a later stage of viral infection, IPCs differentiate into a unique type of mature dendritic cell, which directly regulates the function of T cells and thus links innate and adaptive immune responses. After more than two decades of effort by researchers, IPCs finally claim their place in the hematopoietic chart as the most important cell type in antiviral innate immunity. Understanding IPC biology holds future promise for developing cures for infectious diseases, cancer, and autoimmune diseases.

  16. Trafficking of cell surface beta-amyloid precursor protein: retrograde and transcytotic transport in cultured neurons

    PubMed Central

    1995-01-01

    Amyloid beta-protein (A beta), the principal constituent of senile plaques seen in Alzheimer's disease (AD), is derived by proteolysis from the beta-amyloid precursor protein (beta PP). The mechanism of A beta production in neurons, which are hypothesized to be a rich source of A beta in brain, remains to be defined. In this study, we describe a detailed localization of cell surface beta PP and its subsequent trafficking in primary cultured neurons. Full-length cell surface beta PP was present primarily on perikarya and axons, the latter with a characteristic discontinuous pattern. At growth cones, cell surface beta PP was inconsistently detected. By visualizing the distribution of beta PP monoclonal antibodies added to intact cultures, beta PP was shown to be internalized from distal axons or terminals and retrogradely transported back to perikarya in organelles which colocalized with fluid-phase endocytic markers. Retrograde transport of beta PP was shown in both hippocampal and peripheral sympathetic neurons, the latter using a compartment culture system that isolated cell bodies from distal axons and terminals. In addition, we demonstrated that beta PP from distal axons was transcytotically transported to the surface of perikarya from distal axons in sympathetic neurons. Indirect evidence of this transcytotic pathway was obtained in hippocampal neurons using antisense oligonucleotide to the kinesin heavy chain to inhibit anterograde beta PP transport. Taken together, these results demonstrate novel aspects of beta PP trafficking in neurons, including retrograde axonal transport and transcytosis. Moreover, the axonal predominance of cell surface beta PP is unexpected in view of the recent report of polarized sorting of beta PP to the basolateral domain of MDCK cells. PMID:7721945

  17. Natural Killer Cells Differentiate Human Adipose-Derived Stem Cells and Modulate Their Adipogenic Potential.

    PubMed

    Rezzadeh, Kameron S; Hokugo, Akishige; Jewett, Anahid; Kozlowska, Anna; Segovia, Luis Andres; Zuk, Patricia; Jarrahy, Reza

    2015-09-01

    Natural killer cells are thought to represent more than 30 percent of all lymphocytes within the stromal vascular fraction of lipoaspirates. However, their physiologic interaction with adipocytes and their precursors has never been specifically examined. The authors hypothesized that natural killer cells, by means of cytokine secretion, are capable of promoting the differentiation of adipose-derived stem cells. Human natural killer cells purified from healthy donors' peripheral blood mononuclear cells were activated with a combination of interleukin-2 and anti-CD16 monoclonal antibody; natural killer cell supernatant was collected. Adipose-derived stem cells isolated from raw human lipoaspirates from healthy patients were treated with growth media, growth media with natural killer cell supernatant, adipogenic media, and adipogenic media with natural killer cells supernatant. Flow cytometric analysis was performed on cells using antibodies against B7H1, CD36, CD44, CD34, CD29, and MHC-1. Adipogenic-related gene expression (PPAR-γ, LPL, GPD-1, and aP2) was assessed. Oil Red O staining was performed as a functional assay of adipocyte differentiation and adipogenesis. Adipose-derived stem cells maintained in growth media with natural killer cell supernatant lost markers of "stemness," including CD44, CD34, and CD29; and expressed markers of differentiation, including B7H1 and MHC-1. Adipose-derived stem cells treated with natural killer cell supernatant accumulated small amounts of lipid after 10 days of natural killer cell supernatant treatment. Adipose-derived stem cells treated with natural killer cell supernatant showed altered expression of adipogenesis-associated genes compared with cells maintained in growth media. Adipose-derived stem cells maintained in adipogenic media with natural killer cell supernatant accumulated less lipid than those cells in adipogenic media alone. The authors demonstrate that, through secreted factors, natural killer cells are capable

  18. Biliary tree stem cells, precursors to pancreatic committed progenitors: evidence for possible life-long pancreatic organogenesis.

    PubMed

    Wang, Yunfang; Lanzoni, Giacomo; Carpino, Guido; Cui, Cai-Bin; Dominguez-Bendala, Juan; Wauthier, Eliane; Cardinale, Vincenzo; Oikawa, Tsunekazu; Pileggi, Antonello; Gerber, David; Furth, Mark E; Alvaro, Domenico; Gaudio, Eugenio; Inverardi, Luca; Reid, Lola M

    2013-09-01

    Peribiliary glands (PBGs) in bile duct walls, and pancreatic duct glands (PDGs) associated with pancreatic ducts, in humans of all ages, contain a continuous, ramifying network of cells in overlapping maturational lineages. We show that proximal (PBGs)-to-distal (PDGs) maturational lineages start near the duodenum with cells expressing markers of pluripotency (NANOG, OCT4, and SOX2), proliferation (Ki67), self-replication (SALL4), and early hepato-pancreatic commitment (SOX9, SOX17, PDX1, and LGR5), transitioning to PDG cells with no expression of pluripotency or self-replication markers, maintenance of pancreatic genes (PDX1), and expression of markers of pancreatic endocrine maturation (NGN3, MUC6, and insulin). Radial-axis lineages start in PBGs near the ducts' fibromuscular layers with stem cells and end at the ducts' lumens with cells devoid of stem cell traits and positive for pancreatic endocrine genes. Biliary tree-derived cells behaved as stem cells in culture under expansion conditions, culture plastic and serum-free Kubota's Medium, proliferating for months as undifferentiated cells, whereas pancreas-derived cells underwent only approximately 8-10 divisions, then partially differentiated towards an islet fate. Biliary tree-derived cells proved precursors of pancreas' committed progenitors. Both could be driven by three-dimensional conditions, islet-derived matrix components and a serum-free, hormonally defined medium for an islet fate (HDM-P), to form spheroids with ultrastructural, electrophysiological and functional characteristics of neoislets, including glucose regulatability. Implantation of these neoislets into epididymal fat pads of immunocompromised mice, chemically rendered diabetic, resulted in secretion of human C-peptide, regulatable by glucose, and able to alleviate hyperglycemia in hosts. The biliary tree-derived stem cells and their connections to pancreatic committed progenitors constitute a biological framework for life-long pancreatic

  19. Activating receptor NKG2D targets RAE-1-expressing allogeneic neural precursor cells in a viral model of multiple sclerosis.

    PubMed

    Weinger, Jason G; Plaisted, Warren C; Maciejewski, Sonia M; Lanier, Lewis L; Walsh, Craig M; Lane, Thomas E

    2014-10-01

    Transplantation of major histocompatibility complex-mismatched mouse neural precursor cells (NPCs) into mice persistently infected with the neurotropic JHM strain of mouse hepatitis virus (JHMV) results in rapid rejection that is mediated, in part, by T cells. However, the contribution of the innate immune response to allograft rejection in a model of viral-induced neurological disease has not been well defined. Herein, we demonstrate that the natural killer (NK) cell-expressing-activating receptor NKG2D participates in transplanted allogeneic NPC rejection in mice persistently infected with JHMV. Cultured NPCs derived from C57BL/6 (H-2(b) ) mice express the NKG2D ligand retinoic acid early precursor transcript (RAE)-1 but expression was dramatically reduced upon differentiation into either glia or neurons. RAE-1(+) NPCs were susceptible to NK cell-mediated killing whereas RAE-1(-) cells were resistant to lysis. Transplantation of C57BL/6-derived NPCs into JHMV-infected BALB/c (H-2(d) ) mice resulted in infiltration of NKG2D(+) CD49b(+) NK cells and treatment with blocking antibody specific for NKG2D increased survival of allogeneic NPCs. Furthermore, transplantation of differentiated RAE-1(-) allogeneic NPCs into JHMV-infected BALB/c mice resulted in enhanced survival, highlighting a role for the NKG2D/RAE-1 signaling axis in allograft rejection. We also demonstrate that transplantation of allogeneic NPCs into JHMV-infected mice resulted in infection of the transplanted cells suggesting that these cells may be targets for infection. Viral infection of cultured cells increased RAE-1 expression, resulting in enhanced NK cell-mediated killing through NKG2D recognition. Collectively, these results show that in a viral-induced demyelination model, NK cells contribute to rejection of allogeneic NPCs through an NKG2D signaling pathway. © 2014 AlphaMed Press.

  20. IL-9-Producing Mast Cell Precursors and Food Allergy

    DTIC Science & Technology

    2016-10-01

    development of anaphylactic response to food allergens (This novel cell type was originally named as IMCP9 in the proposal). The central hypothesis is that...transfer Fig. 3 Origin of intestinal MCPs, MMC9, and CD4+TH2 cells in irradiated recipient mice after reconstitution in a murine model of food...producing innate cells with MC-lineage molecular signatures. Bone Marrow MCPs Give Rise to Intestinal MMC9s To examine theMC lineage origin of intestinal

  1. Generation of GABAergic and dopaminergic interneurons from endogenous embryonic olfactory bulb precursor cells.

    PubMed

    Vergaño-Vera, Eva; Yusta-Boyo, María J; de Castro, Fernando; Bernad, Antonio; de Pablo, Flora; Vicario-Abejón, Carlos

    2006-11-01

    During the embryonic period, many olfactory bulb (OB) interneurons arise in the lateral ganglionic eminence (LGE) from precursor cells expressing Dlx2, Gsh2 and Er81 transcription factors. Whether GABAergic and dopaminergic interneurons are also generated within the embryonic OB has not been studied thoroughly. In contrast to abundant Dlx2 and Gsh2 expression in ganglionic eminences (GE), Dlx2 and Gsh2 proteins are not expressed in the E12.5-13.5 mouse OB, whereas the telencephalic pallial domain marker Pax6 is abundant. We found GABAergic and dopaminergic neurons originating from dividing precursor cells in E13.5 OB and in short-term dissociated cultures prepared from the rostral half of E13.5 OB. In OB cultures, 22% of neurons were GAD+, of which 53% were Dlx2+, whereas none expressed Gsh2. By contrast, 70% of GAD+ cells in GE cultures were Dlx2+ and 16% expressed Gsh2. In E13.5 OB slices transplanted with EGFP-labeled E13.5 OB precursor cells, 31.7% of EGFP+ cells differentiated to GABAergic neurons. OB and LGE precursors transplanted into early postnatal OB migrated and differentiated in distinct patterns. Transplanted OB precursors gave rise to interneurons with dendritic spines in close proximity to synaptophysin-positive boutons. Interneurons were also abundant in differentiating OB neural stem cell cultures; the neurons responded to the neurotrophin Bdnf and expressed presynaptic proteins. In vivo, the Bdnf receptor TrkB colocalized with synaptic proteins at the glomeruli. These findings suggest that, in addition to receiving interneurons from the LGE, the embryonic OB contains molecularly distinct local precursor cells that generate mature GABAergic and dopaminergic neurons.

  2. B-cell precursor acute lymphoblastic leukemia and stromal cells communicate through Galectin-3

    PubMed Central

    Fei, Fei; Joo, Eun Ji; Tarighat, Somayeh S.; Schiffer, Isabelle; Paz, Helicia; Fabbri, Muller; Abdel-Azim, Hisham; Groffen, John; Heisterkamp, Nora

    2015-01-01

    The molecular interactions between B-cell precursor acute lymphoblastic leukemia (pre-B ALL) cells and stromal cells in the bone marrow that provide microenvironmentally-mediated protection against therapeutic drugs are not well-defined. Galectin-3 (Lgals3) is a multifunctional galactose-binding lectin with reported location in the nucleus, cytoplasm and extracellular space in different cell types. We previously reported that ALL cells co-cultured with stroma contain high levels of Galectin-3. We here establish that, in contrast to more mature B-lineage cancers, Galectin-3 detected in and on the ALL cells originates from stromal cells, which express it on their surface, secrete it as soluble protein and also in exosomes. Soluble and stromal-bound Galectin-3 is internalized by ALL cells, transported to the nucleus and stimulates transcription of endogenous LGALS3 mRNA. When human and mouse ALL cells develop tolerance to different drugs while in contact with protective stromal cells, Galectin-3 protein levels are consistently increased. This correlates with induction of Galectin-3 transcription in the ALL cells. Thus Galectin-3 sourced from stroma becomes supplemented by endogenous Galectin-3 production in the pre-B ALL cells that are under continuous stress from drug treatment. Our data suggest that stromal Galectin-3 may protect ALL cells through auto-induction of Galectin-3 mRNA and tonic NFκB pathway activation. Since endogenously synthesized Galectin-3 protects pre-B ALL cells against drug treatment, we identify Galectin-3 as one possible target to counteract the protective effects of stroma. PMID:25869099

  3. Retrovirus-mediated conditional immortalization and analysis of established cell lines of osteoclast precursor cells

    SciTech Connect

    Kawata, Shigehisa; Suzuki, Jun; Maruoka, Masahiro; Mizutamari, Megumi; Ishida-Kitagawa, Norihiro; Yogo, Keiichiro; Jat, Parmjit S.; Shishido, Tomoyuki . E-mail: shishid@bs.naist.jp

    2006-11-10

    Osteoclast precursor cells (OPCs) have previously been established from bone marrow cells of SV40 temperature-sensitive T antigen-expressing transgenic mice. Here, we use retrovirus-mediated gene transfer to conditionally immortalize OPCs by expressing temperature-sensitive large T antigen (tsLT) from wild type bone marrow cells. The immortalized OPCs proliferated at the permissive temperature of 33.5 deg. C, but stopped growing at the non-permissive temperature of 39 deg. C. In the presence of receptor activator of NF{kappa}B ligand (RANKL), the OPCs differentiated into tartrate-resistant acid phosphatase (TRAP)-positive cells and formed multinucleate osteoclasts at 33.5 deg. C. From these OPCs, we cloned two types of cell lines. Both differentiated into TRAP-positive cells, but one formed multinucleate osteoclasts while the other remained unfused in the presence of RANKL. These results indicate that the established cell lines are useful for analyzing mechanisms of differentiation, particularly multinucleate osteoclast formation. Retrovirus-mediated conditional immortalization should be a useful method to immortalize OPCs from primary bone marrow cells.

  4. Acute hepatitis A induction of precursor B-cell acute lymphoblastic leukemia: a causal relationship?

    PubMed

    Senadhi, V; Emuron, D; Gupta, R

    2010-09-01

    Precursor B-cell acute lymphoblastic leukemia accounts for 2% of all lymphoid neoplasms in the United States and occurs most frequently in childhood, but can also occur in adults with a median age of 39 years. It is more commonly seen in males and in Caucasians. We present a case of a 51-year-old Caucasian female with the development of precursor B-cell acute lymphoblastic leukemia after suffering acute hepatitis A 4 weeks prior to her diagnosis. She presented with malaise for a month without spontaneous bruising/bleeding, infections, or B-symptoms, such as fevers, night sweats, or unintentional weight loss. Nonspecific viral transformation of bone marrow has been discussed in the literature, but we specifically describe hepatitis A-induced adult-onset precursor B-cell acute lymphoblastic leukemia, which is the first reported case in the literature.

  5. Amyloid precursor protein (APP) and its derivatives change after cellular energy depletion. An in vitro-study.

    PubMed

    Hoyer, A; Bardenheuer, H J; Martin, E; Plaschke, K

    2005-02-01

    To study the relationship between the metabolism of amyloid precursor protein (APP) and cellular energy failure, HEK 293 cells stably transfected with betaAPP 695 underwent graded energy failure induced either by i) hypoxia (pO(2) 25 mm Hg), ii) inhibition of the respiratory chain by sodium azide (NaN(3)), or iii) by combined glucose deprivation/hypoxia of different duration and severity. Secreted APP (APPs) and the derivative betaA4 were quantified autoradiographically by immunoprecipitation, and [(35)S] methionine labeling. APP holoprotein (APPh) was determined by Western blot analysis. The concentrations of the energy-rich metabolites ATP, ADP, creatine phosphate (CrP), and adenosine were measured by high performance liquid chromatography. Mild to moderate energy failure after NaN(3) treatment (2h, 4h) and hypoxia (2h, 8h) was characterized by normal ATP concentration but also by a high reduction in CrP. A stress condition indicated by an increased ATP turnover and adenosine increase was obtained. Intracellular APPh increased but its metabolites APPs and betaA4 as measured in the extracellular compartment decreased. These changes may point to a compensatory response of APP but also to a initial disturbance in intracellular APP metabolism. Severe abnormalities in both energy formation and utilization after 8h NaN(3) and hypoxia glucose deprivation were found to be accompanied by a drastic fall in intracellular APPh concentration by at least 50%, paralleled by an accelerating reduction in the extracellular concentrations of both APPs and betaA4.A significant linear correlation between APPh and ATP and between CrP and betaA4 became obvious. The data of the present study indicate that abnormalities in APP metabolism were generated in an energy-dependent manner. The obvious similarities to sporadic Alzheimer s disease are discussed.

  6. Age-related impairment of T cell-induced skeletal muscle precursor cell function

    PubMed Central

    Dumke, Breanna R.

    2011-01-01

    Sarcopenia is the age-associated loss of skeletal muscle mass and strength. Recent evidence suggests that an age-associated loss of muscle precursor cell (MPC) functionality contributes to sarcopenia. The objectives of the present study were to examine the influence of activated T cells on MPCs and determine whether an age-related defect in this signaling occurs. MPCs were collected from the gastrocnemius and plantaris of 3-mo-old (young) and 32-mo-old (old) animals. Splenic T cells were harvested using anti-CD3 Dynabead isolation. T cells were activated for 48 h with costimulation of 100 IU/ml interleukin-2 (IL-2) and 5 μg/ml of anti-CD28. Costimulation increased 5-bromo-2′-deoxyuridine incorporation of T cells from 13.4 ± 4.6% in control to 64.8 ± 6.0% in costimulated cells. Additionally, T cell cytokines increased proliferation on MPCs isolated from young muscle by 24.0 ± 5.7%, whereas there was no effect on MPCs isolated from aged muscle. T cell cytokines were also found to be a chemoattractant. T cells were able to promote migration of MPCs isolated from young muscle; however, MPCs isolated from aged muscle did not respond to the T cell-released chemokines. Conversely, whereas T cell-released cytokines did not affect myogenesis of MPCs isolated from young animals, there was a decrease in MPCs isolated from old animals. These data suggest that T cells may play a critical role in mediating MPC function. Furthermore, aging may alter T cell-induced MPC function. These findings have implications for developing strategies aimed at increasing MPC migration and proliferation leading to an improved regenerative capacity of aged skeletal muscle. PMID:21325640

  7. Ovary-derived precursor gibberellin A9 is essential for female flower development in cucumber.

    PubMed

    Pimenta Lange, Maria João; Lange, Theo

    2016-12-01

    Gibberellins (GAs) are hormones that control many aspects of plant development, including flowering. It is well known that stamen is the source of GAs that regulate male and bisexual flower development. However, little is known about the role of GAs in female flower development. In cucumber, high levels of GA precursors are present in ovaries and high levels of bioactive GA4 are identified in sepals/petals, reflecting the expression of GA 20-oxidase and 3-oxidase in these organs, respectively. Here, we show that the biologically inactive precursor GA9 moves from ovaries to sepal/petal tissues where it is converted to the bioactive GA4 necessary for female flower development. Transient expression of a catabolic GA 2-oxidase from pumpkin in cucumber ovaries decreases GA9 and GA4 levels and arrests the development of female flowers, and this can be restored by application of GA9 to petals thus confirming its function. Given that bioactive GAs can promote sex reversion of female flowers, movement of biologically inactive precursors, instead of the hormone itself, might help to maintain floral organ identity, ensuring fruit and seed production. © 2016. Published by The Company of Biologists Ltd.

  8. Prolonged cultivation of hippocampal neural precursor cells shifts their differentiation potential and selects for aneuploid cells.

    PubMed

    Nguyen, The Duy; Widera, Darius; Greiner, Johannes; Müller, Janine; Martin, Ina; Slotta, Carsten; Hauser, Stefan; Kaltschmidt, Christian; Kaltschmidt, Barbara

    2013-12-01

    Neural precursor cells (NPCs) are lineage-restricted neural stem cells with limited self-renewal, giving rise to a broad range of neural cell types such as neurons, astrocytes, and oligodendrocytes. Despite this developmental potential, the differentiation capacity of NPCs has been controversially discussed concerning the trespassing lineage boundaries, for instance resulting in hematopoietic competence. Assessing their in vitro plasticity, we isolated nestin+/Sox2+, NPCs from the adult murine hippocampus. In vitro-expanded adult NPCs were able to form neurospheres, self-renew, and differentiate into neuronal, astrocytic, and oligodendrocytic cells. Although NPCs cultivated in early passage efficiently gave rise to neuronal cells in a directed differentiation assay, extensively cultivated NPCs revealed reduced potential for ectodermal differentiation. We further observed successful differentiation of long-term cultured NPCs into osteogenic and adipogenic cell types, suggesting that NPCs underwent a fate switch during culture. NPCs cultivated for more than 12 passages were aneuploid (abnormal chromosome numbers such as 70 chromosomes). Furthermore, they showed growth factor-independent proliferation, a hallmark of tumorigenic transformation. In conclusion, our findings substantiate the lineage restriction of NPCs from adult mammalian hippocampus. Prolonged cultivation results, however, in enhanced differentiation potential, which may be attributed to transformation events leading to aneuploid cells.

  9. Decreased demand for olfactory periglomerular cells impacts on neural precursor cell viability in the rostral migratory stream

    PubMed Central

    Langenfurth, Anika; Gu, Song; Bautze, Verena; Zhang, Caiyi; Neumann, Julia E.; Schüller, Ulrich; Stock, Kristin; Wolf, Susanne A.; Maier, Anna-Maria; Mastrella, Giorgia; Pak, Andrew; Cheng, Hongwei; Kälin, Roland E.; Holmbeck, Kenn; Strotmann, Jörg; Kettenmann, Helmut; Glass, Rainer

    2016-01-01

    The subventricular zone (SVZ) provides a constant supply of new neurons to the olfactory bulb (OB). Different studies have investigated the role of olfactory sensory input to neural precursor cell (NPC) turnover in the SVZ but it was not addressed if a reduced demand specifically for periglomerular neurons impacts on NPC-traits in the rostral migratory stream (RMS). We here report that membrane type-1 matrix metalloproteinase (MT1-MMP) deficient mice have reduced complexity of the nasal turbinates, decreased sensory innervation of the OB, reduced numbers of olfactory glomeruli and reduced OB-size without alterations in SVZ neurogenesis. Large parts of the RMS were fully preserved in MT1-MMP-deficient mice, but we detected an increase in cell death-levels and a decrease in SVZ-derived neuroblasts in the distal RMS, as compared to controls. BrdU-tracking experiments showed that homing of NPCs specifically to the glomerular layer was reduced in MT1-MMP-deficient mice in contrast to controls while numbers of tracked cells remained equal in other OB-layers throughout all experimental groups. Altogether, our data show the demand for olfactory interneurons in the glomerular layer modulates cell turnover in the RMS, but has no impact on subventricular neurogenesis. PMID:27573347

  10. Export of Precursor tRNAIle from the Nucleus to the Cytoplasm in Human Cells

    PubMed Central

    Wei, Min; Zhao, Xia; Liu, Mi; Niu, Meijuan; Seif, Elias; Kleiman, Lawrence

    2016-01-01

    In the current concept, tRNA maturation in vertebrate cells, including splicing of introns, trimming of 5’ leader and 3’ trailer, and adding of CCA, is thought to occur exclusively in the nucleus. Here we provide evidence to challenge this concept. Unspliced intron-containing precursor tRNAIle was identified in Human Immunodeficiency Virus type 1 (HIV-1) virions, which are synthesized in the cytoplasm. Northern blot, confocal microscopy and quantitative RT-PCR further verified enrichment of this unspliced tRNAIle within the cytoplasm in human cells. In addition to containing an intron, the cytoplasmic precursor tRNAIle also contains a short incompletely processed 5´ leader and a 3´ trailer, which abundance is around 1000 fold higher than the nuclear precursor tRNAIle with long 5’ leader and long 3’ trailer. In vitro data also suggest that the cytoplasmic unspliced end-immature precursor tRNAIle could be processed by short isoform of RNase Z, but not long isoform of RNase Z. These data suggest that precursor tRNAs could export from the nucleus to the cytoplasm in human cells, instead of be processed only in the nucleus. PMID:27101286

  11. Export of Precursor tRNAIle from the Nucleus to the Cytoplasm in Human Cells.

    PubMed

    Wei, Min; Zhao, Xia; Liu, Mi; Niu, Meijuan; Seif, Elias; Kleiman, Lawrence

    2016-01-01

    In the current concept, tRNA maturation in vertebrate cells, including splicing of introns, trimming of 5' leader and 3' trailer, and adding of CCA, is thought to occur exclusively in the nucleus. Here we provide evidence to challenge this concept. Unspliced intron-containing precursor tRNAIle was identified in Human Immunodeficiency Virus type 1 (HIV-1) virions, which are synthesized in the cytoplasm. Northern blot, confocal microscopy and quantitative RT-PCR further verified enrichment of this unspliced tRNAIle within the cytoplasm in human cells. In addition to containing an intron, the cytoplasmic precursor tRNAIle also contains a short incompletely processed 5´ leader and a 3´ trailer, which abundance is around 1000 fold higher than the nuclear precursor tRNAIle with long 5' leader and long 3' trailer. In vitro data also suggest that the cytoplasmic unspliced end-immature precursor tRNAIle could be processed by short isoform of RNase Z, but not long isoform of RNase Z. These data suggest that precursor tRNAs could export from the nucleus to the cytoplasm in human cells, instead of be processed only in the nucleus.

  12. Hematopoietic reconstitution by multipotent adult progenitor cells: precursors to long-term hematopoietic stem cells

    PubMed Central

    Serafini, Marta; Dylla, Scott J.; Oki, Masayuki; Heremans, Yves; Tolar, Jakub; Jiang, Yuehua; Buckley, Shannon M.; Pelacho, Beatriz; Burns, Terry C.; Frommer, Sarah; Rossi, Derrick J.; Bryder, David; Panoskaltsis-Mortari, Angela; O'Shaughnessy, Matthew J.; Nelson-Holte, Molly; Fine, Gabriel C.; Weissman, Irving L.; Blazar, Bruce R.; Verfaillie, Catherine M.

    2007-01-01

    For decades, in vitro expansion of transplantable hematopoietic stem cells (HSCs) has been an elusive goal. Here, we demonstrate that multipotent adult progenitor cells (MAPCs), isolated from green fluorescent protein (GFP)-transgenic mice and expanded in vitro for >40–80 population doublings, are capable of multilineage hematopoietic engraftment of immunodeficient mice. Among MAPC-derived GFP+CD45.2+ cells in the bone marrow of engrafted mice, HSCs were present that could radioprotect and reconstitute multilineage hematopoiesis in secondary and tertiary recipients, as well as myeloid and lymphoid hematopoietic progenitor subsets and functional GFP+ MAPC-derived lymphocytes that were functional. Although hematopoietic contribution by MAPCs was comparable to control KTLS HSCs, approximately 103-fold more MAPCs were required for efficient engraftment. Because GFP+ host-derived CD45.1+ cells were not observed, fusion is not likely to account for the generation of HSCs by MAPCs. PMID:17227908

  13. Redox changes induced in hippocampal precursor cells by heavy ion irradiation.

    PubMed

    Limoli, C L; Giedzinski, E; Baure, J; Rola, R; Fike, J R

    2007-06-01

    Hippocampal precursors retain the capacity to proliferate and differentiate throughout life, and their progeny, immature neurons, can undergo neurogenesis, a process believed to be important in maintaining the cognitive health of an organism. A variety of stresses including irradiation have been shown to deplete neural precursor cells, an effect that inhibits neurogenesis and is associated with the onset of cognitive impairments. Our past work has shown that neural precursor cells exposed to X-rays or protons exhibit a prolonged increase in oxidative stress, a factor we hypothesize to be critical in regulating the function of these cells after irradiation and other stresses. Here we report that irradiation of hippocampal precursor cells with high-linear energy transfer (LET) 1 GeV/nucleon 56Fe ions leads to significantly higher levels of oxidative stress when compared to lower LET radiations (X-rays, protons). Irradiation with 1 Gy of 56Fe ions elicits twofold to fivefold higher levels of reactive oxygen species (ROS) compared to unirradiated controls, and at lower doses (precursors exhibit a linear dose response 6 h after heavy ion exposure. The use of the antioxidant lipoic acid (LA) was able to reduce ROS levels below background levels when added before or after 56Fe ion irradiation. These results conclusively show that low doses of 56Fe ions can elicit significant levels of oxidative stress in neural precursor cells. Given the prevalence of heavy ions in space and the duration of interplanetary travel, these data suggest that astronauts are at risk for developing cognitive decrements. However, our results also indicate that antioxidants delivered before as radioprotective agents or after as mitigating agents hold promise as effective countermeasures for ameliorating certain adverse effects of heavy ion exposure to the CNS.

  14. Poly cyclic aromatic hydrocarbons in Recent lake sediments—II. Compounds derived from biogenic precursors during early diagenesis

    NASA Astrophysics Data System (ADS)

    Wakeham, Stuart G.; Schaffner, Christian; Giger, Walter

    1980-03-01

    Five groups of polycyclic aromatic hydrocarbons (PAH) thought to be derived by early-diagenetic transformations of biogenic precursors are apparently present in Recent sediments of four lakes (Lake Lucerne. Lake Zürich, and Greifensee. Switzerland, and Lake Washington, northwest U.S.A.) These natural PAH include: (1) perylene. (2) an extended series of phenanthrene homologs. (3) retene and pimanthrene derived from diterpenes. (4) a series of tetra- and pentacyclic PAH derived from pentacyclic triterpenes of the amyrin-type. (5) tetra- and pentacyclic PAH formed from pentacyclic triterpenes with five-membered E-rings. Since these PAH are abundant in very young sediment layers, the transformation reactions involved appear to be faster than previously thought and may be microbially mediated. There is no evidence that anthropogenic or petrogenic sources can account for the distributions of these groups of PAH in cores of Recent lake sediments.

  15. Isolation of Precursor Cells from Waste Solid Fat Tissue

    NASA Technical Reports Server (NTRS)

    Byerly, Diane; Sognier, Marguerite A.

    2009-01-01

    A process for isolating tissue-specific progenitor cells exploits solid fat tissue obtained as waste from such elective surgical procedures as abdominoplasties (tummy tucks) and breast reductions. Until now, a painful and risky process of aspiration of bone marrow has been used to obtain a limited number of tissue- specific progenitor cells. The present process yields more tissue-specific progenitor cells and involves much less pain and risk for the patient. This process includes separation of fat from skin, mincing of the fat into small pieces, and forcing a fat saline mixture through a sieve. The mixture is then digested with collagenase type I in an incubator. After centrifugation tissue-specific progenitor cells are recovered and placed in a tissue-culture medium in flasks or Petri dishes. The tissue-specific progenitor cells can be used for such purposes as (1) generating three-dimensional tissue equivalent models for studying bone loss and muscle atrophy (among other deficiencies) and, ultimately, (2) generating replacements for tissues lost by the fat donor because of injury or disease.

  16. Nestin Reporter Transgene Labels Multiple Central Nervous System Precursor Cells

    PubMed Central

    Walker, Avery S.; Goings, Gwendolyn E.; Kim, Yongsoo; Miller, Richard J.; Chenn, Anjen; Szele, Francis G.

    2010-01-01

    Embryonic neuroepithelia and adult subventricular zone (SVZ) stem and progenitor cells express nestin. We characterized a transgenic line that expresses enhanced green fluorescent protein (eGFP) specified to neural tissue by the second intronic enhancer of the nestin promoter that had several novel features. During embryogenesis, the dorsal telencephalon contained many and the ventral telencephalon few eGFP+ cells. eGFP+ cells were found in postnatal and adult neurogenic regions. eGFP+ cells in the SVZ expressed multiple phenotype markers, glial fibrillary acidic protein, Dlx, and neuroblast-specific molecules suggesting the transgene is expressed through the lineage. eGFP+ cell numbers increased in the SVZ after cortical injury, suggesting this line will be useful in probing postinjury neurogenesis. In non-neurogenic regions, eGFP was strongly expressed in oligodendrocyte progenitors, but not in astrocytes, even when they were reactive. This eGFP+ mouse will facilitate studies of proliferative neuroepithelia and adult neurogenesis, as well as of parenchymal oligodendrocytes. PMID:21527990

  17. Can manipulation of differentiation conditions eliminate proliferative cells from a population of ES cell-derived forebrain cells?

    PubMed Central

    Precious, Sophie V.; Kelly, Claire M.; Allen, Nicholas D.; Rosser, Anne E.

    2016-01-01

    ABSTRACT There is preliminary evidence that implantation of primary fetal striatal cells provides functional benefit in patients with Huntington's disease, a neurodegenerative condition resulting in loss of medium-sized spiny neurons (MSN) of the striatum. Scarcity of primary fetal tissue means it is important to identify a renewable source of cells from which to derive donor MSNs. Embryonic stem (ES) cells, which predominantly default to telencephalic-like precursors in chemically defined medium (CDM), offer a potentially inexhaustible supply of cells capable of generating the desired neurons. Using an ES cell line, with the forebrain marker FoxG1 tagged to the LacZ reporter, we assessed effects of known developmental factors on the yield of forebrain-like precursor cells in CDM suspension culture. Addition of FGF2, but not DKK1, increased the proportion of FoxG1-expressing cells at day 8 of neural induction. Oct4 was expressed at day 8, but was undetectable by day 16. Differentiation of day 16 precursors generated GABA-expressing neurons, with few DARPP32 positive MSNs. Transplantation of day 8 precursor cells into quinolinic acid-lesioned striata resulted in generation of teratomas. However, transplantation of day 16 precursors yielded grafts expressing neuronal markers including NeuN, calbindin and parvalbumin, but no DARPP32 6 weeks post-transplantation. Manipulation of fate of ES cells requires optimization of both concentration and timing of addition of factors to culture systems to generate the desired phenotypes. Furthermore, we highlight the value of increasing the precursor phase of ES cell suspension culture when directing differentiation toward forebrain fate, so as to dramatically reduce the risk of teratoma formation. PMID:27606335

  18. Early postradition recovery of hematopoietic stromal precursor cells

    SciTech Connect

    Todriya, T.V.

    1985-04-01

    The aim of this investigation was an immunohistochemical study of alpha-endorphin-producing cells and also a study of rat mast cells (MC in the antral mucosa of the human stomach. Men aged 18 to 30 years undergoing in-patient treatment wre studied. According to the results of radioimmunoassay, antibodies against alpha-endorphin did not react with enkephalins, beta-endorphin, or the C-terminal fragment of beta-endorphin, but had cross reactivity of about 10% with gammaendorphin. Results were subjected to statistical analysis by Student's test at a 85% level of significance and they are shown. The facts presented here suggest that MC of human gastric mucosa include argyrophilic cells which contain alpha-endorphin.

  19. Detection of Biosynthetic Precursors, Discovery of Glycosylated Forms, and Homeostasis of Calcitonin in Human Cancer Cells.

    PubMed

    Cao, Feihua; Gamble, Allan B; Onagi, Hideki; Howes, Joanna; Hennessy, James E; Gu, Chen; Morgan, Jeremy A M; Easton, Christopher J

    2017-07-05

    The peptide hormone calcitonin is intimately connected with human cancer development and proliferation. Its biosynthesis is reasoned to proceed via glycine-, α-hydroxyglycine-, glycyllysine-, and glycyllysyllysine-extended precursors; however, as a result of the limitations of current analytical methods, until now, there has been no procedure capable of detecting these individual species in cell or tissue samples. Therefore, their presence and dynamics in cancer had not been established. Here, we report the first methodology for the separation, detection, and quantification of calcitonin and each of its precursors in human cancer cells. We also report the discovery and characterization of O-glycosylated calcitonin and its analogous biosynthetic precursors. Through direct and simultaneous analysis of the glycosylated and nonglycosylated species, we interrogate the hormone biosynthesis. This shows that the cellular calcitonin level is maintained to mitigate effects of biosynthetic enzyme inhibitors that substantially change the proportions of calcitonin-related species released into the culture medium.

  20. Effect of ionizing radiation on human skeletal muscle precursor cells

    PubMed Central

    Jurdana, Mihaela; Cemazar, Maja; Pegan, Katarina; Mars, Tomaz

    2013-01-01

    Background Long term effects of different doses of ionizing radiation on human skeletal muscle myoblast proliferation, cytokine signalling and stress response capacity were studied in primary cell cultures. Materials and methods Human skeletal muscle myoblasts obtained from muscle biopsies were cultured and irradiated with a Darpac 2000 X-ray unit at doses of 4, 6 and 8 Gy. Acute effects of radiation were studied by interleukin – 6 (IL-6) release and stress response detected by the heat shock protein (HSP) level, while long term effects were followed by proliferation capacity and cell death. Results Compared with non-irradiated control and cells treated with inhibitor of cell proliferation Ara C, myoblast proliferation decreased 72 h post-irradiation, this effect was more pronounced with increasing doses. Post-irradiation myoblast survival determined by measurement of released LDH enzyme activity revealed increased activity after exposure to irradiation. The acute response of myoblasts to lower doses of irradiation (4 and 6 Gy) was decreased secretion of constitutive IL-6. Higher doses of irradiation triggered a stress response in myoblasts, determined by increased levels of stress markers (HSPs 27 and 70). Conclusions Our results show that myoblasts are sensitive to irradiation in terms of their proliferation capacity and capacity to secret IL-6. Since myoblast proliferation and differentiation are a key stage in muscle regeneration, this effect of irradiation needs to be taken in account, particularly in certain clinical conditions. PMID:24294183

  1. 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...

  2. 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...

  3. Hedgehog signalling stimulates precursor cell accumulation and impairs epithelial maturation in the murine oesophagus.

    PubMed

    van Dop, Willemijn A; Rosekrans, Sanne L; Uhmann, Anja; Jaks, Viljar; Offerhaus, G Johan A; van den Bergh Weerman, Marius A; Kasper, Maria; Heijmans, Jarom; Hardwick, James C H; Verspaget, Hein W; Hommes, Daan W; Toftgård, Rune; Hahn, Heidi; van den Brink, Gijs R

    2013-03-01

    In the intestine Hedgehog (Hh) signalling is directed from epithelium to mesenchyme and negatively regulates epithelial precursor cell fate. The role of Hh signalling in the oesophagus has not been studied in vivo. Here the authors examined the role of Hh signalling in epithelial homeostasis of oesophagus. The authors used transgenic mice in which the Hh receptor Patched1 (Ptch1) could be conditionally inactivated in a body-wide manner and mice in which Gli1 could be induced specifically in the epithelium of the skin and oesophagus. Effects on epithelial homeostasis of the oesophagus were examined using immunohistochemistry, in situ hybridisation, transmission electron microscopy and real-time PCR. Hh signalling was examined in patients with oesophageal squamous cell carcinoma (SCC) by quantitative real-time PCR. Sonic Hh is signalled in an autocrine manner in the basal layer of the oesophagus. Activation of Hh signalling resulted in an expansion of the epithelial precursor cell compartment and failure of epithelial maturation and migration. Levels of Hh targets GLI1, HHIP and PTCH1 were increased in SCC compared with normal tissue from the same patients. Here the authors find that Hh signalling positively regulates the precursor cell compartment in the oesophageal epithelium in an autocrine manner. Since Hh signalling targets precursor cells in the oesophageal epithelium and signalling is increased in SCCs, Hh signalling may be involved in oesophageal SCC formation.

  4. Alpha-ketoglutarate Curbs Differentiation and Induces Cell Death in Mesenchymal Stromal Precursors with Mitochondrial Dysfunction.

    PubMed

    Singh, Karmveer; Krug, Linda; Basu, Abhijit; Meyer, Patrick; Treiber, Nicolai; Vander Beken, Seppe; Wlaschek, Meinhard; Kochanek, Stefan; Bloch, Wilhelm; Geiger, Hartmut; Maity, Pallab; Scharffetter-Kochanek, Karin

    2017-04-11

    Increased concentrations of reactive oxygen species (ROS) originating from dysfunctional mitochondria contribute to diverse aging-related degenerative disorders. But so far little is known about the impact of distinct ROS on metabolism and fate of stromal precursor cells. We here demonstrate that an increase in superoxide anion radicals due to superoxide dismutase 2 (Sod2) deficiency in stromal precursor cells suppress osteogenic and adipogenic differentiation through fundamental changes in the global metabolite landscape. Our data identify impairment of the pyruvate and L-glutamine metabolism causing toxic accumulation of alpha-ketoglutarate in the Sod2 deficient and intrinsically aged stromal precursor cells as a major cause for their reduced lineage differentiation. Alpha-ketoglutarate accumulation led to enhanced nucleocytoplasmic vacuolation and chromatin condensation-mediated cell death in Sod2 deficient stromal precursor cells as a consequence of DNA damage, Hif-1α instability and reduced histone H3 (Lys27) acetylation. These findings hold promise for prevention and treatment of mitochondrial disorders commonly associated with aged individuals. This article is protected by copyright. All rights reserved.

  5. Sonic hedgehog-expressing basal cells are general post-mitotic precursors of functional taste receptor cells

    PubMed Central

    Miura, Hirohito; Scott, Jennifer K.; Harada, Shuitsu; Barlow, Linda A.

    2014-01-01

    Background Taste buds contain ~60 elongate cells and several basal cells. Elongate cells comprise three functional taste cell types: I - glial cells, II - bitter/sweet/umami receptor cells, and III - sour detectors. Although taste cells are continuously renewed, lineage relationships among cell types are ill-defined. Basal cells have been proposed as taste bud stem cells, a subset of which express Sonic hedgehog (Shh). However, Shh+ basal cells turnover rapidly suggesting that Shh+ cells are precursors of some or all taste cell types. Results To fate map Shh-expressing cells, mice carrying ShhCreERT2 and a high (CAG-CAT-EGFP) or low (R26RLacZ) efficiency reporter allele were given tamoxifen to activate Cre in Shh+ cells. Using R26RLacZ, lineage-labeled cells occur singly within buds, supporting a post-mitotic state for Shh+ cells. Using either reporter, we show that Shh+ cells differentiate into all three taste cell types, in proportions reflecting cell type ratios in taste buds (I > II > III). Conclusions Shh+ cells are not stem cells, but are post-mitotic, immediate precursors of taste cells. Shh+ cells differentiate into each of the three taste cell types, and the choice of a specific taste cell fate is regulated to maintain the proper ratio within buds. PMID:24590958

  6. Identification of embryonic precursor cells that differentiate into thymic epithelial cells expressing autoimmune regulator

    PubMed Central

    Takizawa, Nobukazu; Miyauchi, Maki; Yanai, Hiromi; Tateishi, Ryosuke; Shinzawa, Miho; Yoshinaga, Riko; Kurihara, Masaaki; Yasuda, Hisataka; Sakamoto, Reiko; Yoshida, Nobuaki

    2016-01-01

    Medullary thymic epithelial cells (mTECs) expressing autoimmune regulator (Aire) are critical for preventing the onset of autoimmunity. However, the differentiation program of Aire-expressing mTECs (Aire+ mTECs) is unclear. Here, we describe novel embryonic precursors of Aire+ mTECs. We found the candidate precursors of Aire+ mTECs (pMECs) by monitoring the expression of receptor activator of nuclear factor-κB (RANK), which is required for Aire+ mTEC differentiation. pMECs unexpectedly expressed cortical TEC molecules in addition to the mTEC markers UEA-1 ligand and RANK and differentiated into mTECs in reaggregation thymic organ culture. Introduction of pMECs in the embryonic thymus permitted long-term maintenance of Aire+ mTECs and efficiently suppressed the onset of autoimmunity induced by Aire+ mTEC deficiency. Mechanistically, pMECs differentiated into Aire+ mTECs by tumor necrosis factor receptor-associated factor 6-dependent RANK signaling. Moreover, nonclassical nuclear factor-κB activation triggered by RANK and lymphotoxin-β receptor signaling promoted pMEC induction from progenitors exhibiting lower RANK expression and higher CD24 expression. Thus, our findings identified two novel stages in the differentiation program of Aire+ mTECs. PMID:27401343

  7. Identification of embryonic precursor cells that differentiate into thymic epithelial cells expressing autoimmune regulator.

    PubMed

    Akiyama, Nobuko; Takizawa, Nobukazu; Miyauchi, Maki; Yanai, Hiromi; Tateishi, Ryosuke; Shinzawa, Miho; Yoshinaga, Riko; Kurihara, Masaaki; Demizu, Yosuke; Yasuda, Hisataka; Yagi, Shintaro; Wu, Guoying; Matsumoto, Mitsuru; Sakamoto, Reiko; Yoshida, Nobuaki; Penninger, Josef M; Kobayashi, Yasuhiro; Inoue, Jun-Ichiro; Akiyama, Taishin

    2016-07-25

    Medullary thymic epithelial cells (mTECs) expressing autoimmune regulator (Aire) are critical for preventing the onset of autoimmunity. However, the differentiation program of Aire-expressing mTECs (Aire(+) mTECs) is unclear. Here, we describe novel embryonic precursors of Aire(+) mTECs. We found the candidate precursors of Aire(+) mTECs (pMECs) by monitoring the expression of receptor activator of nuclear factor-κB (RANK), which is required for Aire(+) mTEC differentiation. pMECs unexpectedly expressed cortical TEC molecules in addition to the mTEC markers UEA-1 ligand and RANK and differentiated into mTECs in reaggregation thymic organ culture. Introduction of pMECs in the embryonic thymus permitted long-term maintenance of Aire(+) mTECs and efficiently suppressed the onset of autoimmunity induced by Aire(+) mTEC deficiency. Mechanistically, pMECs differentiated into Aire(+) mTECs by tumor necrosis factor receptor-associated factor 6-dependent RANK signaling. Moreover, nonclassical nuclear factor-κB activation triggered by RANK and lymphotoxin-β receptor signaling promoted pMEC induction from progenitors exhibiting lower RANK expression and higher CD24 expression. Thus, our findings identified two novel stages in the differentiation program of Aire(+) mTECs.

  8. Oxidative stress affects processing of amyloid precursor protein in vascular endothelial cells.

    PubMed

    Muche, Abebe; Arendt, Thomas; Schliebs, Reinhard

    2017-01-01

    Oxidative stress is thought to be a key player in the pathogenesis of neurodegenerative dementia, including Alzheimer's disease (AD). It has been assumed that oxidative stress contributes to the ß-amyloid deposition in cerebral blood vessels. In order to prove this hypothesis, we examined the effect of oxidative stress on the processing of amyloid precursor protein (APP) in primary endothelial cells (EC) derived from cerebral cortical tissue of transgenic Tg2576 mice. Following exposure of EC by 1 μM hydrogen peroxide for up to 48 hours, formation and secretion of APP cleavage products sAPPα and sAPPß into the culture medium as well as the expression of endothelial APP were assessed. Oxidative stress resulted in enhanced secretion of sAPPß into the culture medium as compared to controls (absence of hydrogen peroxide), which was accompanied by an increased APP expression, induction of VEGF synthesis, nitric oxide and oxygen free radicals productions, and differential changes of endothelial phospo-p42/44 MAPK expression. The data suggest that oxidative stress may represent a major risk factor in causing Aß deposition in the brain vascular system by initiating the amyloidogenic route of endothelial APP processing. The enhanced β-secretase activity following oxidative stress exposure, possibly promoted by phosphorylation of p42/44 MAPK.

  9. Recruitment of myeloid but not endothelial precursor cells facilitates tumor re-growth after local irradiation

    PubMed Central

    Kozin, Sergey V.; Kamoun, Walid S.; Huang, Yuhui; Dawson, Michelle R.; Jain, Rakesh K.; Duda, Dan G.

    2010-01-01

    Tumor neovascularization and growth may be promoted by recruitment of bone marrow-derived cells (BMDCs), which include endothelial precursor cells (EPCs) and “vascular modulatory” myelomonocytic (CD11b+) cells. BMDCs may also drive tumor re-growth after certain chemotherapeutic and vascular disruption treatments. In this study, we evaluated the role of BMDC recruitment in breast and lung carcinoma xenograft models after local irradiation (LI). We depleted the bone marrow by including whole body irradiation (WBI) of 6Gy as part of a total tumor dose of 21Gy, and compared the growth delay with the one achieved after LI of 21Gy. In both models, including WBI induced longer tumor growth delays. Moreover, including WBI increased lung tumor control probability by LI. Exogenous delivery of BMDCs from radiation-naïve donors partially abrogated the WBI effect. Myeloid BMDCs, primarily macrophages, rapidly accumulated in tumors after LI. Intratumoral expression of SDF-1α, a chemokine that promotes tissue retention of BMDCs, was noted 2 days after LI. Conversely, treatment with an inhibitor of SDF-1α receptor CXCR4 (AMD3100) with LI significantly delayed tumor re-growth. However, when administered starting from 5 days post-LI, AMD3100 treatment was ineffective. Lastly, with restorative bone marrow transplantation of Tie2-GFP-labeled BMDC population we observed an increased number of monocytes but not EPCs in tumors that recurred following LI. Our results suggest that an increase in intratumoral SDF-1α triggered by local irradiation recruits myelomonocyte/macrophage which promote tumor re-growth. PMID:20631066

  10. Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages

    PubMed Central

    Froehlich, Jacob Michael; Seiliez, Iban; Gabillard, Jean-Charles; Biga, Peggy R.

    2014-01-01

    Due to the inherent difficulty and time involved with studying the myogenic program in vivo, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata, however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e. teleost fish) and full regeneration following appendage loss (i.e. urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4. PMID:24835774

  11. Chemically Diverse Toxicants Converge on Fyn and c-Cbl to Disrupt Precursor Cell Function

    PubMed Central

    Li, Zaibo; Dong, Tiefei; Pröschel, Chris; Noble, Mark

    2007-01-01

    Identification of common mechanistic principles that shed light on the action of the many chemically diverse toxicants to which we are exposed is of central importance in understanding how toxicants disrupt normal cellular function and in developing more effective means of protecting against such effects. Of particular importance is identifying mechanisms operative at environmentally relevant toxicant exposure levels. Chemically diverse toxicants exhibit striking convergence, at environmentally relevant exposure levels, on pathway-specific disruption of receptor tyrosine kinase (RTK) signaling required for cell division in central nervous system (CNS) progenitor cells. Relatively small toxicant-induced increases in oxidative status are associated with Fyn kinase activation, leading to secondary activation of the c-Cbl ubiquitin ligase. Fyn/c-Cbl pathway activation by these pro-oxidative changes causes specific reductions, in vitro and in vivo, in levels of the c-Cbl target platelet-derived growth factor receptor-α and other c-Cbl targets, but not of the TrkC RTK (which is not a c-Cbl target). Sequential Fyn and c-Cbl activation, with consequent pathway-specific suppression of RTK signaling, is induced by levels of methylmercury and lead that affect large segments of the population, as well as by paraquat, an organic herbicide. Our results identify a novel regulatory pathway of oxidant-mediated Fyn/c-Cbl activation as a shared mechanism of action of chemically diverse toxicants at environmentally relevant levels, and as a means by which increased oxidative status may disrupt mitogenic signaling. These results provide one of a small number of general mechanistic principles in toxicology, and the only such principle integrating toxicology, precursor cell biology, redox biology, and signaling pathway analysis in a predictive framework of broad potential relevance to the understanding of pro-oxidant–mediated disruption of normal development. PMID:17298174

  12. Antimicrobial Activity of Artemisinin and Precursor Derived from In Vitro Plantlets of Artemisia annua L.

    PubMed Central

    Appalasamy, Suganthi; Lo, Kiah Yann; Ch'ng, Song Jin; Nornadia, Ku; Othman, Ahmad Sofiman; Chan, Lai-Keng

    2014-01-01

    Artemisia annua L., a medicinal herb, produces secondary metabolites with antimicrobial property. In Malaysia due to the tropical hot climate, A. annua could not be planted for production of artemisinin, the main bioactive compound. In this study, the leaves of three in vitro A. annua L. clones were, extracted and two bioactive compounds, artemisinin and a precursor, were isolated by thin layer chromatography. These compounds were found to be effective in inhibiting the growth of Gram-positive and Gram-negative bacteria but not Candida albicans. Their antimicrobial activity was similar to that of antibactericidal antibiotic streptomycin. They were found to inhibit the growth of the tested microbes at the minimum inhibition concentration of 0.09 mg/mL, and toxicity test using brine shrimp showed that even the low concentration of 0.09 mg/mL was very lethal towards the brine shrimps with 100% mortality rate. This study hence indicated that in vitro cultured plantlets of A. annua can be used as the alternative method for production of artemisinin and its precursor with antimicrobial activities. PMID:24575401

  13. Retinoic acid regulates the development of oligodendrocyte precursor cells in vitro.

    PubMed

    Laeng, P; Décimo, D; Pettmann, B; Janet, T; Labourdette, G

    1994-12-15

    Cultures of oligodendrocyte precursor cells can be grown from brain hemispheres of newborn rats. These cells, also called O-2A progenitor cells, can differentiate in vitro into oligodendrocytes or type 2 astrocytes. Basic FGF and PDGF are known to stimulate their proliferation and delay their differentiation. Lack or excess of retinoic acid (RA) has been known for a long time to alter brain development suggesting that this compound is involved in normal brain development. Here we report that RA partially inhibits both the proliferation and the differentiation of oligodendrocyte precursor cells. It also down-regulates the mitogenic effect of bFGF on these cells while keeping them in an immature stage. RA is more effective than bFGF in inhibiting myelin basic protein mRNA expression in these cells, and like bFGF, it preserves their bipotential character. RA nuclear receptors RAR-alpha and their transcripts are expressed in oligodendrocyte precursor cells as seen by Western blot, Northern blot and in situ hybridization. The expression of RAR-alpha transcripts is stimulated transiently by RA alone or associated to bFGF. The expression of RAR-beta transcripts is not constitutive and is induced by RA alone or associated to bFGF and to a lesser extent by bFGF alone. These results suggest that retinoids participate in the control of the development of glial cells of the oligodendrocyte lineage.

  14. Plasma cell precursors: long-distance travelers looking for a home.

    PubMed

    Luther, Sanjiv A

    2010-07-23

    Little is known about the migration of plasma cell precursors to the lymph node medulla. In this issue of Immunity, Fooksman et al. (2010) propose that this migration is largely independent of chemotactic cues but follows a long linear walk of random orientation.

  15. Monocyte-Derived Suppressor Cells in Transplantation.

    PubMed

    Ochando, Jordi; Conde, Patricia; Bronte, Vincenzo

    Myeloid-derived suppressor cells (MDSC) are cells of myeloid origin with enhanced suppressive function. They are negative regulators of the immune responses and comprise a heterogeneous mixture of immunosuppressive cells of monocytic (M-MDSC) and granulocytic (G-MDSC) origin. A more recent nomenclature proposes the term "suppressive monocyte derived cells" (suppressive MCs) to define CSF1/CSF2-dependent mouse suppressor cells that develop from common monocyte progenitors (cMoPs) after birth. Here, we review the literature about monocytic-derived cells with demonstrated suppressor function in vitro and in vivo within the context of solid organ transplantation.

  16. The Mesenchymal Precursor Cell Marker Antibody STRO-1 Binds to Cell Surface Heat Shock Cognate 70.

    PubMed

    Fitter, Stephen; Gronthos, Stan; Ooi, Soo Siang; Zannettino, Andrew C W

    2016-12-27

    Since its discovery more than 25 years ago, the STRO-1 antibody has played a fundamental role in defining the hierarchical nature of mesenchymal precursor cells (MPC) and their progeny. STRO-1 antibody binding remains a hallmark of immature pluripotent MPC. Despite the significance of STRO-1 in the MPC field, the identity of the antigen has remained elusive. Using a combination of two-dimensional gel electrophoresis, coupled with Western blotting and Tandem mass spectroscopy, we have identified the STRO-1 antigen as heat shock cognate 70 (HSC70;HSPA8). STRO-1 binds to immune-precipitated HSC70 and siRNA-mediated knock down of HSPA8 reduced STRO-1 binding. STRO-1 surface binding does not correlate with HSC70 expression and sequestration of cholesterol reduces STRO-1 surface binding, suggesting that the plasma membrane lipid composition may be an important determinant in the presentation of HSC70 on the cell surface. HSC70 is present on the surface of STRO-1(+) but not STRO-1(-) cell lines as assessed by cell surface biotinylation and recombinant HSC70 blocks STRO-1 binding to the cell surface. The STRO-1 epitope on HSC70 was mapped to the ATPase domain using a series of deletion mutants in combination with peptide arrays. Deletion of the first four amino acids of the consensus epitope negated STRO-1 binding. Notably, in addition to HSC70, STRO-1 cross-reacts with heat shock protein 70 (HSP70), however all the clonogenic cell activity is restricted to the STRO-1(BRIGHT) /HSP70(-) fraction. These results provide important insight into the properties that define multipotent MPC and provide the impetus to explore the role of cell surface HSC70 in MPC biology. Stem Cells 2016.

  17. The adult CNS retains the potential to direct region-specific differentiation of a transplanted neuronal precursor cell line.

    PubMed

    Shihabuddin, L S; Hertz, J A; Holets, V R; Whittemore, S R

    1995-10-01

    The chronic survival and differentiation of the conditionally immortalized neuronal cell line, RN33B, was examined following transplantation into the adult and neonatal rat hippocampus and cerebral cortex. In clonal culture, differentiated RN33B cells express p75NTR and trkB mRNA and protein, and respond to brain-derived neurotrophic factor treatment by inducing c-fos mRNA. Transplanted cells, identified using immunohistochemistry to detect beta-galactosidase expression, were seen in most animals up to 24 weeks posttransplantation (the latest time point examined). Stably integrated cells with various morphologies consistent with their transplantation site were observed. In the cerebral cortex, many RN33B cells differentiated with morphologies similar to pyramidal neurons and stellate cells. In the hippocampal formation, many RN33B cells assumed morphologies similar to pyramidal neurons characteristic of CA1 and CA3 regions, granular cell layer neurons of the dentate gyrus, and polymorphic neurons of the hilar region. Identical morphologies were observed in both adult and neonatal hosts, although a greater percentage of beta-galactosidase immunoreactive cells had differentiated in the neonatal brains. These results suggest that RN33B cells have the developmental plasticity to respond to local microenvironmental signals and that the adult brain retains the capacity to direct the differentiation of neuronal precursor cells in a direction that is consistent with that of endogenous neurons.

  18. Naive CD8⁺ T-cell precursors display structured TCR repertoires and composite antigen-driven selection dynamics.

    PubMed

    Neller, Michelle A; Ladell, Kristin; McLaren, James E; Matthews, Katherine K; Gostick, Emma; Pentier, Johanne M; Dolton, Garry; Schauenburg, Andrea J A; Koning, Dan; Fontaine Costa, Ana Isabel C A; Watkins, Thomas S; Venturi, Vanessa; Smith, Corey; Khanna, Rajiv; Miners, Kelly; Clement, Mathew; Wooldridge, Linda; Cole, David K; van Baarle, Debbie; Sewell, Andrew K; Burrows, Scott R; Price, David A; Miles, John J

    2015-08-01

    Basic parameters of the naive antigen (Ag)-specific T-cell repertoire in humans remain poorly defined. Systematic characterization of this 'ground state' immunity in comparison with memory will allow a better understanding of clonal selection during immune challenge. Here, we used high-definition cell isolation from umbilical cord blood samples to establish the baseline frequency, phenotype and T-cell antigen receptor (TCR) repertoire of CD8(+) T-cell precursor populations specific for a range of viral and self-derived Ags. Across the board, these precursor populations were phenotypically naive and occurred with hierarchical frequencies clustered by Ag specificity. The corresponding patterns of TCR architecture were highly ordered and displayed partial overlap with adult memory, indicating biased structuring of the T-cell repertoire during Ag-driven selection. Collectively, these results provide new insights into the complex nature and dynamics of the naive T-cell compartment.

  19. Sonic hedgehog acts cell-autonomously on muscle precursor cells to generate limb muscle diversity

    PubMed Central

    Anderson, Claire; Williams, Victoria C.; Moyon, Benjamin; Daubas, Philippe; Tajbakhsh, Shahragim; Buckingham, Margaret E.; Shiroishi, Toshihiko; Hughes, Simon M.; Borycki, Anne-Gaëlle

    2012-01-01

    How muscle diversity is generated in the vertebrate body is poorly understood. In the limb, dorsal and ventral muscle masses constitute the first myogenic diversification, as each gives rise to distinct muscles. Myogenesis initiates after muscle precursor cells (MPCs) have migrated from the somites to the limb bud and populated the prospective muscle masses. Here, we show that Sonic hedgehog (Shh) from the zone of polarizing activity (ZPA) drives myogenesis specifically within the ventral muscle mass. Shh directly induces ventral MPCs to initiate Myf5 transcription and myogenesis through essential Gli-binding sites located in the Myf5 limb enhancer. In the absence of Shh signaling, myogenesis is delayed, MPCs fail to migrate distally, and ventral paw muscles fail to form. Thus, Shh production in the limb ZPA is essential for the spatiotemporal control of myogenesis and coordinates muscle and skeletal development by acting directly to regulate the formation of specific ventral muscles. PMID:22987640

  20. Electrical stimulation by enzymatic biofuel cell to promote proliferation, migration and differentiation of muscle precursor cells.

    PubMed

    Lee, Jae Ho; Jeon, Won-Yong; Kim, Hyug-Han; Lee, Eun-Jung; Kim, Hae-Won

    2015-01-01

    Electrical stimulation is a very important biophysical cue for skeletal muscle maintenance and myotube formation. The absence of electrical signals from motor neurons causes denervated muscles to atrophy. Herein, we investigate for the first time the utility of an enzymatic biofuel cell (EBFC) as a promising means for mimicking native electrical stimulation. EBFC was set up using two different enzymes: one was glucose oxidase (GOX) used for the generation of anodic current followed by the oxidation of glucose; the other was Bilirubin oxidase (BOD) for the generation of cathodic current followed by the reduction of oxygen. We studied the behaviors of muscle precursor cells (MPCs) in terms of proliferation, migration and differentiation under different electrical conditions. The EBFC electrical stimulations significantly increased cell proliferation and migration. Furthermore, the electrical stimulations promoted the differentiation of cells into myotube formation based on expressions at the gene and protein levels. The EBFC set up, with its free forms adjustable to any implant design, was subsequently applied to the nanofiber scaffolding system. The MPCs were demonstrated to be stimulated in a similar manner as the 2D culture conditions, suggesting potential applications of the EBFC system for muscle repair and regeneration.

  1. Sonic Hedgehog Controls the Phenotypic Fate and Therapeutic Efficacy of Grafted Neural Precursor Cells in a Model of Nigrostriatal Neurodegeneration.

    PubMed

    Madhavan, Lalitha; Daley, Brian F; Davidson, Beverly L; Boudreau, Ryan L; Lipton, Jack W; Cole-Strauss, Allyson; Steece-Collier, Kathy; Collier, Timothy J

    2015-01-01

    The expression of soluble growth and survival promoting factors by neural precursor cells (NPCs) is suggested to be a prominent mechanism underlying the protective and regenerative effects of these cells after transplantation. Nevertheless, how and to what extent specific NPC-expressed factors contribute to therapeutic effects is not well understood. Using RNA silencing, the current study investigated the roles of two donor NPC molecules, namely glial cell-line derived neurotrophic factor (GDNF) and sonic hedgehog (SHH), in the protection of substantia nigra dopamine neurons in rats treated with 6-hydroxydopamine (6-OHDA). Analyses indicate that as opposed to the knock-down of GDNF, SHH inhibition caused a profound decline in nigrostriatal neuroprotection. Further, SHH silencing also curbed endogenous neurogenesis and the migration of host brdU+/dcx+ neural precursors into the striatum, which was present in the animals receiving control or GDNF silenced NPCs. A change in graft phenotype, mainly reflected by a reduced proportion of undifferentiated nestin+ cells, as well as a significantly greater host microglial activity, suggested an important role for these processes in the attenuation of neuroprotection and neurogenesis upon SHH silencing. Overall these studies reveal core mechanisms fundamental to grafted NPC-based therapeutic effects, and delineate the particular contributions of two graft-expressed molecules, SHH and GDNF, in mediating midbrain dopamine neuron protection, and host plasticity after NPC transplantation.

  2. Allogeneic Mesenchymal Precursor Cell Therapy to Limit Remodeling After Myocardial Infarction: The Effect of Cell Dosage

    PubMed Central

    Hamamoto, Hirotsugu; Gorman, Joseph H.; Ryan, Liam P.; Hinmon, Robin; Martens, Timothy P.; Schuster, Michael D.; Plappert, Theodore; Kiupel, Matti; St. John-Sutton, Martin G.; Itescu, Silviu; Gorman, Robert C.

    2011-01-01

    Background This experiment assessed the dose-dependent effect of a unique allogeneic STRO-3–positive mesenchymal precursor cell (MPC) on postinfarction left ventricular (LV) remodeling. The MPCs were administered in a manner that would simulate an off-the-self, early postinfarction, preventative approach to cardiac cell therapy in a sheep transmural myocardial infarct (MI) model. Methods Allogeneic MPCs were isolated from male crossbred sheep. Forty-six female sheep underwent coronary ligation to produce a transmural LV anteroapical infarction. One hour after infarction, the borderzone myocardium received an injection of 25, 75, 225, or 450 × 106 MPCs, or cell medium. Echocardiography was performed at 4 and 8 weeks after MI to quantify LV end-diastolic (LVEDV) and end-systolic volumes (LVESV), ejection fraction (EF), and infarct expansion. CD31 and smooth muscle actin (SMA) immunohistochemical staining was performed on infarct and borderzone specimens to quantify vascular density. Results Compared with controls, low-dose (25 and 75 × 106 cells) MPC treatment significantly attenuated infarct expansion and increases in LVEDV and LVESV. EF was improved at all cell doses. CD31 and SMA immunohistochemical staining demonstrated increased vascular density in the borderzone only at the lower cell doses. There was no evidence of myocardial regeneration within the infarct. Conclusion Allogeneic STRO-3 positive MPCs attenuate the remodeling response to transmural MI in a clinically relevant large-animal model. This effect is associated with vasculogenesis and arteriogenesis within the borderzone and infarct and is most pronounced at lower cell doses. PMID:19231391

  3. Waste polyvinylchloride derived pitch as a precursor to develop carbon fibers and activated carbon fibers.

    PubMed

    Qiao, W M; Yoon, S H; Mochida, I; Yang, J H

    2007-01-01

    Polyvinylchloride (PVC) was successfully recycled through the solvent extraction from waste pipe with an extraction yield of ca. 86%. The extracted PVC was pyrolyzed by a two-stage process (260 and 410 degrees C) to obtain free-chlorine PVC based pitch through an effective removal of chlorine from PVC during the heat-treatment. As-prepared pitch (softening point: 220 degrees C) was spun, stabilized, carbonized into carbon fibers (CFs), and further activated into activated carbon fibers (ACFs) in a flow of CO2. As-prepared CFs show comparable mechanical properties to commercial CFs, whose maximum tensile strength and modulus are 862 MPa and 62 GPa, respectively. The resultant ACFs exhibit a high surface area of 1200 m2/g, narrow pore size distribution and a low oxygen content of 3%. The study provides an effective insight to recycle PVC from waste PVC and develop a carbon precursor for high performance carbon materials such as CFs and ACFs.

  4. Subretinal transplantation of MACS purified photoreceptor precursor cells into the adult mouse retina.

    PubMed

    Eberle, Dominic; Santos-Ferreira, Tiago; Grahl, Sandra; Ader, Marius

    2014-02-22

    Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to

  5. Esophageal squamous cell carcinoma - precursor lesions and early diagnosis

    PubMed Central

    Lopes, Antonio Barros; Fagundes, Renato Borges

    2012-01-01

    Squamous cell carcinoma of the esophagus (SCCE) carries a poor prognosis due to late diagnosis. Early detection is highly desirable, since surgical and endoscopic resection offers the only possible cure for esophageal cancer. Population screening should be undertaken in high risk areas, and in low or moderate risk areas for people with risk factors (alcoholics, smokers, mate drinkers, history of head and neck cancer, achalasia and lye stricture of the esophagus). Esophageal balloon cytology is an easy and inexpensive sampling technique, but the current methods are insufficient for primary screening due to sampling errors. Conventional endoscopy with biopsy remains the standard procedure for the identification of pre-malignant and early malignant changes in esophageal mucosa and endoscopic detection. It may be enhanced by several techniques such as dye and optic chromoendoscopy, magnifying endoscopy, and optical-based spectroscopic and imaging modalities. Since more than 80% of SCCE deaths occur in developing countries, where expensive techniques such as narrow band imaging (NBI) and autofluorescence imaging are unavailable, the most cost-effective tool for targeting biopsies may be Lugol dye chromoendoscopy, since it is easy, accurate, inexpensive and available worldwide. In ideal conditions, or in developed countries, is it reasonable to think that optimal detection will require a combination of techniques, such as the combination of Lugol’s chromoendoscopy and NBI to identify esophageal areas that require further characterization by a high resolution technique. The efficacy and cost-effectiveness will determine whether these modalities will become part of standard endoscopy practice. PMID:22267978

  6. HIV-1 broadly neutralizing antibody precursor B cells revealed by germline-targeting immunogen

    SciTech Connect

    Jardine, Joseph G.; Kulp, Daniel W.; Havenar-Daughton, Colin; Sarkar, Anita; Briney, Bryan; Sok, Devin; Sesterhenn, Fabian; Ereno-Orbea, June; Kalyuzhniy, Oleksandr; Deresa, Isaiah; Hu, Xiaozhen; Spencer, Skye; Jones, Meaghan; Georgeson, Erik; Adachi, Yumiko; Kubitz, Michael; deCamp, Allan C.; Julien, Jean -Philippe; Wilson, Ian A.; Burton, Dennis R.; Crotty, Shane; Schief, William R.

    2016-03-25

    Induction of broadly neutralizing antibodies (bnAbs) is a major HIV vaccine goal. Germline-targeting immunogens aim to initiate bnAb induction by activating bnAb germline precursor B cells. Critical unmet challenges are to determine whether bnAb precursor naïve B cells bind germline-targeting immunogens and occur at sufficient frequency in humans for reliable vaccine responses. We employed deep mutational scanning and multi-target optimization to develop a germline-targeting immunogen (eOD-GT8) for diverse VRC01-class bnAbs. We then used the immunogen to isolate VRC01-class precursor naïve B cells from HIV-uninfected donors. Frequencies of true VRC01-class precursors, their structures, and their eOD-GT8 affinities support this immunogen as a candidate human vaccine prime. Lastly, these methods could be applied to germline targeting for other classes of HIV bnAbs and for Abs to other pathogens.

  7. HIV-1 broadly neutralizing antibody precursor B cells revealed by germline-targeting immunogen

    DOE PAGES

    Jardine, Joseph G.; Kulp, Daniel W.; Havenar-Daughton, Colin; ...

    2016-03-25

    Induction of broadly neutralizing antibodies (bnAbs) is a major HIV vaccine goal. Germline-targeting immunogens aim to initiate bnAb induction by activating bnAb germline precursor B cells. Critical unmet challenges are to determine whether bnAb precursor naïve B cells bind germline-targeting immunogens and occur at sufficient frequency in humans for reliable vaccine responses. We employed deep mutational scanning and multi-target optimization to develop a germline-targeting immunogen (eOD-GT8) for diverse VRC01-class bnAbs. We then used the immunogen to isolate VRC01-class precursor naïve B cells from HIV-uninfected donors. Frequencies of true VRC01-class precursors, their structures, and their eOD-GT8 affinities support this immunogen asmore » a candidate human vaccine prime. Lastly, these methods could be applied to germline targeting for other classes of HIV bnAbs and for Abs to other pathogens.« less

  8. Interaction of type A lantibiotics with undecaprenol-bound cell envelope precursors.

    PubMed

    Müller, Anna; Ulm, Hannah; Reder-Christ, Katrin; Sahl, Hans-Georg; Schneider, Tanja

    2012-06-01

    Lantibiotics are a unique group within the antimicrobial peptides characterized by the presence of thioether amino acids (lanthionine and methyllanthionine). These peptides are produced by and primarily act on Gram-positive bacteria exerting multiple activities at the cytoplasmic membrane of susceptible strains. Previously, the cell wall precursor lipid II was identified as the molecular target for the prototype lantibiotic nisin. Binding and sequestration of lipid II blocks the incorporation of the central cell wall precursor into the growing peptidoglycan network, thereby inhibiting the formation of a functional cell wall. Additionally, nisin combines this activity with a unique target-mediated pore formation, using lipid II as a docking molecule. The interaction with the pyrophosphate moiety of lipid II is crucial for nisin binding. We show that, besides binding to lipid II, nisin interacts with the lipid intermediates lipid III (undecaprenol-pyrophosphate-N-acetyl-glucosamine) and lipid IV (undecaprenol-pyrophosphate-N-acetyl-glucosamine-N-acetyl-mannosamine) of the wall teichoic acid (WTA) biosynthesis pathway. Binding of nisin to the precursors was observed at a stoichiometry of 2:1. The specific interaction with WTA precursors further promoted target-mediated pore formation in artificial lipid bilayers. Specific interactions with lipid III and lipid IV could also be demonstrated for related type A lantibiotics, for example, gallidermin, containing the conserved lipid-II-binding motif.

  9. HIV-1 broadly neutralizing antibody precursor B cells revealed by germline-targeting immunogen.

    PubMed

    Jardine, Joseph G; Kulp, Daniel W; Havenar-Daughton, Colin; Sarkar, Anita; Briney, Bryan; Sok, Devin; Sesterhenn, Fabian; Ereño-Orbea, June; Kalyuzhniy, Oleksandr; Deresa, Isaiah; Hu, Xiaozhen; Spencer, Skye; Jones, Meaghan; Georgeson, Erik; Adachi, Yumiko; Kubitz, Michael; deCamp, Allan C; Julien, Jean-Philippe; Wilson, Ian A; Burton, Dennis R; Crotty, Shane; Schief, William R

    2016-03-25

    Induction of broadly neutralizing antibodies (bnAbs) is a major HIV vaccine goal. Germline-targeting immunogens aim to initiate bnAb induction by activating bnAb germline precursor B cells. Critical unmet challenges are to determine whether bnAb precursor naïve B cells bind germline-targeting immunogens and occur at sufficient frequency in humans for reliable vaccine responses. Using deep mutational scanning and multitarget optimization, we developed a germline-targeting immunogen (eOD-GT8) for diverse VRC01-class bnAbs. We then used the immunogen to isolate VRC01-class precursor naïve B cells from HIV-uninfected donors. Frequencies of true VRC01-class precursors, their structures, and their eOD-GT8 affinities support this immunogen as a candidate human vaccine prime. These methods could be applied to germline targeting for other classes of HIV bnAbs and for Abs to other pathogens.

  10. Generation, Release, and Uptake of the NAD Precursor Nicotinic Acid Riboside by Human Cells.

    PubMed

    Kulikova, Veronika; Shabalin, Konstantin; Nerinovski, Kirill; Dölle, Christian; Niere, Marc; Yakimov, Alexander; Redpath, Philip; Khodorkovskiy, Mikhail; Migaud, Marie E; Ziegler, Mathias; Nikiforov, Andrey

    2015-11-06

    NAD is essential for cellular metabolism and has a key role in various signaling pathways in human cells. To ensure proper control of vital reactions, NAD must be permanently resynthesized. Nicotinamide and nicotinic acid as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR) are the major precursors for NAD biosynthesis in humans. In this study, we explored whether the ribosides NR and NAR can be generated in human cells. We demonstrate that purified, recombinant human cytosolic 5'-nucleotidases (5'-NTs) CN-II and CN-III, but not CN-IA, can dephosphorylate the mononucleotides nicotinamide mononucleotide and nicotinic acid mononucleotide (NAMN) and thus catalyze NR and NAR formation in vitro. Similar to their counterpart from yeast, Sdt1, the human 5'-NTs require high (millimolar) concentrations of nicotinamide mononucleotide or NAMN for efficient catalysis. Overexpression of FLAG-tagged CN-II and CN-III in HEK293 and HepG2 cells resulted in the formation and release of NAR. However, NAR accumulation in the culture medium of these cells was only detectable under conditions that led to increased NAMN production from nicotinic acid. The amount of NAR released from cells engineered for increased NAMN production was sufficient to maintain viability of surrounding cells unable to use any other NAD precursor. Moreover, we found that untransfected HeLa cells produce and release sufficient amounts of NAR and NR under normal culture conditions. Collectively, our results indicate that cytosolic 5'-NTs participate in the conversion of NAD precursors and establish NR and NAR as integral constituents of human NAD metabolism. In addition, they point to the possibility that different cell types might facilitate each other's NAD supply by providing alternative precursors.

  11. Selective beta-cell differentiation of dissociated embryonic pancreatic precursor cells cultured in synthetic polyethylene glycol hydrogels.

    PubMed

    Mason, Mariah N; Mahoney, Melissa J

    2009-06-01

    Continuing advances in islet cell transplantation have been promising; however, several limitations, including severe shortage of transplantable islets, hinder the widespread use of this therapy. Pancreatic precursor cells are one alternative to cadaveric donor islets. These cells found in the developing pancreatic buds are capable of self-renewal and also have the innate ability to become insulin-producing beta-cells. For this work, bioinert polyethylene glycol (PEG) hydrogels were chosen as the supportive three-dimensional matrix for encapsulation of dissociated pancreatic precursor cells obtained from the dorsal pancreatic bud of day-15 rat embryos. This culture system was selected in order to eliminate cell-extracellular matrix and cell-cell signal heterogeneity present when intact pancreatic buds are embedded in protein-based gels, the typical in vitro culture conditions used to study this cell population. In this study it was found that (1) dissociated precursor cells maintain a robust viability for 7 days in PEG hydrogel culture, (2) encapsulated cells selectively differentiate into insulin-expressing beta-cells, and (3) differentiated beta-cells have releasable insulin stores, but are not achieving a mature, glucose responsive phenotype. These findings suggest that encapsulating dissociated pancreatic precursor cells in an environment designed to minimize the heterogeneous signaling cues present during development or in standard culture conditions generates a population highly enriched in pancreatic beta-cells; however, future efforts must focus on achieving glucose responsiveness in this cell population. Further, these results indicate that differentiation down a beta-cell lineage may be the default pathway in pancreatic development.

  12. Tripartite containing motif 32 modulates proliferation of human neural precursor cells in HIV-1 neurodegeneration

    PubMed Central

    Fatima, M; Kumari, R; Schwamborn, J C; Mahadevan, A; Shankar, S K; Raja, R; Seth, P

    2016-01-01

    In addition to glial cells, HIV-1 infection occurs in multipotent human neural precursor cells (hNPCs) and induces quiescence in NPCs. HIV-1 infection of the brain alters hNPC stemness, leading to perturbed endogenous neurorestoration of the CNS following brain damage by HIV-1, compounding the severity of dementia in adult neuroAIDS cases. In pediatric neuroAIDS cases, HIV-1 infection of neural stem cell can lead to delayed developmental milestones and impaired cognition. Using primary cultures of human fetal brain-derived hNPCs, we gained novel insights into the role of a neural stem cell determinant, tripartite containing motif 32 (TRIM32), in HIV-1 Tat-induced quiescence of NPCs. Acute HIV-1 Tat treatment of hNPCs resulted in proliferation arrest but did not induce differentiation. Cellular localization and levels of TRIM32 are critical regulators of stemness of NPCs. HIV-1 Tat exposure increased nuclear localization and levels of TRIM32 in hNPCs. The in vitro findings were validated by studying TRIM32 localization and levels in frontal cortex of HIV-1-seropositive adult patients collected at post mortem as well as by infection of hNPCs by HIV-1. We observed increased percentage of cells with nuclear localization of TRIM32 in the subventricular zone (SVZ) as compared with age-matched controls. Our quest for probing into the mechanisms revealed that TRIM32 is targeted by miR-155 as downregulation of miR-155 by HIV-1 Tat resulted in upregulation of TRIM32 levels. Furthermore, miR-155 or siRNA against TRIM32 rescued HIV-1 Tat-induced quiescence in NPCs. Our findings suggest a novel molecular cascade involving miR-155 and TRIM32 leading to HIV-1 Tat-induced attenuated proliferation of hNPCs. The study also uncovered an unidentified role for miR-155 in modulating human neural stem cell proliferation, helping in better understanding of hNPCs and diseased brain. PMID:26586575

  13. Glial commitment of mesencephalic neural precursor cells expanded as neurospheres precludes their engagement in niche-dependent dopaminergic neurogenesis.

    PubMed

    Baizabal, José-Manuel; Cano-Martínez, Agustina; Valencia, Concepción; Santa-Olalla, Jesús; Young, Kaylene M; Rietze, Rodney L; Bartlett, Perry F; Covarrubias, Luis

    2012-05-01

    Neural precursor cells (NPCs) with high proliferative potential are commonly expanded in vitro as neurospheres. As a population, neurosphere cells show long-term self-renewal capacity and multipotentiality in vitro. These features have led to the assumption that neurosphere cells represent an expansion of the endogenous NPCs residing within the embryonic and adult brain. If this is the case, in principle, bona-fide expansion of endogenous NPCs should not significantly affect their capacity to respond to their original niche of differentiation. To address this issue, we generated primary neurospheres from the dopaminergic niche of the ventral mesencephalon and then transplanted these cells to their original niche within mesencephalic explant cultures. Primary neurosphere cells showed poor capacity to generate dopaminergic neurons in the mesencephalic niche of dopaminergic neurogenesis. Instead, most primary neurosphere cells showed glial commitment as they differentiated into astrocytes in an exclusively neurogenic niche. Subculture of primary cells demonstrated that the neurosphere assay does not amplify niche-responsive dopaminergic progenitors. Further, neurospheres cells were largely unable to acquire the endogenous positional identity within the Nkx6.1(+), Nkx2.2(+), and Pax7(+) domains of mesencephalic explants. Finally, we demonstrate that our observations are not specific for embryonic mesencephalic cells, as NPCs in the adult subventricular zone also showed an intrinsic fate switch from neuronal to glial potential upon neurosphere amplification. Our data suggest that neurosphere formation does not expand the endogenous neurogenic NPCs but rather promotes amplification of gliogenic precursors that do not respond to niche-derived signals of cellular specification and differentiation.

  14. Patient-Derived Antibody Targets Tumor Cells

    Cancer.gov

    An NCI Cancer Currents blog on an antibody derived from patients that killed tumor cells in cell lines of several cancer types and slowed tumor growth in mouse models of brain and lung cancer without evidence of side effects.

  15. Proteomics Applications in Dental Derived Stem Cells.

    PubMed

    Li, Jie; Tian, Weidong; Song, Jinlin

    2017-07-01

    At present, the existence of a variety of dental derived stem cells has been documented. These cells displayed promising clinical application potential not only for teeth and its surrounding tissue regeneration, but also for other tissues, such as nerve and bone regeneration. Proteomics is an unbiased, global informatics tool that provides information on all protein expression levels as well as post-translational modification in cells or tissues and is applicable to dental derived stem cells research. Over the last decade, considerable progress has been made to study the global proteome, secrotome, and membrane proteome of dental derived stem cells. Here, we present an overview of the proteomics studies in the context of stem cell research. Particular attention is given to dental derived stem cell types as well as current challenges and opportunities. J. Cell. Physiol. 232: 1602-1610, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  16. Functional determination of the differentiation potential of ventral mesencephalic neural precursor cells during dopaminergic neurogenesis.

    PubMed

    Guerrero-Flores, Gilda; Bastidas-Ponce, Aimée; Collazo-Navarrete, Omar; Guerra-Crespo, Magdalena; Covarrubias, Luis

    2017-09-01

    The ventral mesencephalic neural precursor cells (vmNPCs) that give rise to dopaminergic (DA) neurons have been identified by the expression of distinct genes (e.g., Lmx1a, Foxa2, Msx1/2). However, the commitment of these NPCs to the mesencephalic DA neuronal fate has not been functionally determined. Evaluation of the plasticity of vmNPCs suggests that their commitment occurs after E10.5. Here we show that E9.5 vmNPCs implanted in an ectopic area of E10.5 mesencephalic explants, retained their specification marker Lmx1a and efficiently differentiated into neurons but did not express the gene encoding tyrosine hydroxylase (Th), the limiting enzyme for dopamine synthesis. A proportion of E10.5-E11.5 implanted vmNPCs behaved as committed, deriving into Th(+) neurons in ectopic sites. Interestingly, implanted cells from E12.5 embryos were unable to give rise to a significant number of Th(+) neurons. Concomitantly, differentiation assays in culture and in mesencephalic explants treated with Fgf2+LIF detected vmNPCs with astrogenic potential since E11.5. Despite this, a full suspension of E12.5 vmNPCs give rise to DA neurons in a similar proportion as those of E10.5 when they were transplanted into adult brain, but astrocytes were only detected with the former population. These data suggest that the subventricular postmitotic progenitors present in E12.5 ventral mesencephalon are unable to implant in embryonic explants and are the source of DA neurons in the transplanted adult brain. Based on our findings we propose that during DA differentiation committed vmNPCs emerge at E10.5 and they exhaust their neurogenic capacity with the rise of NPCs with astrogenic potential. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Neurogenesis and precursor cell differences in the dorsal and ventral adult canine hippocampus.

    PubMed

    Lowe, Aileen; Dalton, Marshall; Sidhu, Kuldip; Sachdev, Perminder; Reynolds, Brent; Valenzuela, Michael

    2015-04-23

    During evolution a unique anterior-posterior flexure posited the canine dentate gyrus in two distinct dorsal and ventral positions. We therefore sought to explore neurogenesis and neurogenic cell-related difference along the canine hippocampal dorsal-ventral axis. Post mortem histological analysis revealed 49.1% greater doublecortin (DCX)-positive cells and a 158.5% greater percentage of double labeled DCX-positive/neuronal nuclei (NeuN) positive cells in the dorsal subgranular zone compared to the ventral. We then show neural precursor cells isolated from fresh hippocampal tissue are capable of proliferating long term, and after differentiation, express neuronal and glial markers. Dorsal hippocampal isolates produced a 120.0% higher frequency of sphere-forming neural precursor cells compared to ventral hippocampal tissue. Histological DCX and neurosphere assay results were highly correlated. Overall, we provide the first evidence that the dorsal canine hippocampus has a markedly higher rate of adult neurogenesis than the ventral hippocampus, possibly related to a greater frequency of contributory neural precursor cells.

  18. A new precursor for the immobilization of enzymes inside sol-gel-derived hybrid silica nanocomposites containing polysaccharides.

    PubMed

    Shchipunov, Yurii A; Karpenko, Tat'yana Yu; Bakunina, Irina Yu; Burtseva, Yuliya V; Zvyagintseva, Tat'yana N

    2004-01-30

    Tetrakis(2-hydroxyethyl) orthosilicate (THEOS) introduced by Hoffmann et al. (J. Phys. Chem. B., 106 (2002) 1528) was first used to prepare hybrid nanocomposites containing various polysaccharides and immobilize enzymes in these materials. Two different types of O-glycoside hydrolyses (EC3.2.1), 1-->3-beta-D-glucanase LIV from marine mollusk Spisula sacchalinensis and alpha-D-galactosidase from marine bacterium Pseudoalteromonas sp. KMM 701, were taken for the immobilization. To reveal whether the polysaccharide inside the hybrid material influences the enzyme entrapment and functioning, negatively charged xanthan, cationic derivative of hydroxyethylcellulose and uncharged locust bean gum were examined. The mechanical properties of these nanocomposites were characterized by a dynamic rheology and their structure by a scanning electron microscopy. It was found that 1-->3-beta-D-glucanase was usually immobilized without the loss of its activity, while the alpha-D-galactosidase activity in the immobilized state depended on the polysaccharide type of material. An important point is that the amount of immobilized enzymes was small, comparable to their content in the living cells. It was shown by the scanning electron microscopy that the hybrid nanocomposites are sufficiently porous that allows the enzymatic substrates and products to diffuse from an external aqueous solution to the enzymes, whereas protein molecules were immobilized firmly and not easily washed out of the silica matrix. A sharp increase of the enzyme lifetime (more than a hundred times) was observed after the immobilization. As established, the efficient entrapment of enzymes is caused by few advantages of new precursor over the currently used TEOS and TMOS: (i) organic solvents and catalysts are not needed owing to the complete solubility of THEOS in water and the catalytic effect of polysaccharides on the sol-gel processes; (ii) the entrapment of enzymes can be performed at any pH which is suitable

  19. Differentiation of functionally active mouse T lymphocytes from functionally inactive bone marrow precursors II. Limited recovery of T-cell responses from mouse bone marrow in tissue culture.

    PubMed

    Gorczynski, M; MacRae, S

    1977-11-01

    The limited differentiation of mature T cell function from mouse bone marrow in tissue culture is described and compared with similar differentiation occuring in vivo in irradiated bone marrow protected mice. Data are presented to show that a pool of precursors, similar in size to that able to produce early (transient?) regeneration in thymectomized recipients, is responsible for the development of mitogen responsive T cells active in MLC (proliferation) and CML (development of cytotoxic cells) assays. In contrast, a helper cell population which augments antibody formation from T-depleted normal spleen cells derives from a pool of similar precursors yet does not seem to be theta positive. Similarly, larger cells (perhaps typical of those giving rise to suppressor T cells in vivo) give rise to a suppressor cell pool after 4 days of culture, though again only a fraction of this suppressor activity could be attributed to theta positive cells. It is suggested that much of the data for regenration of T lymphocytes in vitro from T-depleted sources needs to be re-interpreted in terms of this evidence for a pool of post-thymic precursors of T cells in such T-deficient cell populations.

  20. Differentiation of functionally active mouse T lymphocytes from functionally inactive bone marrow precursors II. Limited recovery of T-cell responses from mouse bone marrow in tissue culture.

    PubMed Central

    Gorczynski, M; MacRae, S

    1977-01-01

    The limited differentiation of mature T cell function from mouse bone marrow in tissue culture is described and compared with similar differentiation occuring in vivo in irradiated bone marrow protected mice. Data are presented to show that a pool of precursors, similar in size to that able to produce early (transient?) regeneration in thymectomized recipients, is responsible for the development of mitogen responsive T cells active in MLC (proliferation) and CML (development of cytotoxic cells) assays. In contrast, a helper cell population which augments antibody formation from T-depleted normal spleen cells derives from a pool of similar precursors yet does not seem to be theta positive. Similarly, larger cells (perhaps typical of those giving rise to suppressor T cells in vivo) give rise to a suppressor cell pool after 4 days of culture, though again only a fraction of this suppressor activity could be attributed to theta positive cells. It is suggested that much of the data for regenration of T lymphocytes in vitro from T-depleted sources needs to be re-interpreted in terms of this evidence for a pool of post-thymic precursors of T cells in such T-deficient cell populations. PMID:304032

  1. The roles of Frizzled-3 and Wnt3a on melanocyte development: in vitro studies on neural crest cells and melanocyte precursor cell lines.

    PubMed

    Chang, Chung-Hsing; Tsai, Rong-Kung; Tsai, Ming-Hsien; Lin, Yi-Hsiung; Hirobe, Tomohisa

    2014-08-01

    Wnt3a and Frizzled-3 are both expressed in the dorsal neural tube that gives rise to the neural crest in Xenopus, zebrafish and mice. Melanocytes originate from the neural crest (NC) and postnatally, melanocyte stem cells reside in the hair follicle bulge and in the dermis. However, the roles of Wnt3a and Frizzled-3 in melanocyte development have not been clarified. The aim of this study was to delineate the expression of Frizzled-3 in murine melanocyte lineage and human melanocytes, and to study the effects of Wnt3a on melanocyte development at various stages. Murine NC explant cultures and three NC-derived melanocyte lineage cell lines, including NCCmelb4M5 (Kit(-) melanocyte precursors), NCCmelb4 (Kit(+) melanoblasts) and NCCmelan5 (differentiated melanocytes), and human epidermal melanocytes were treated with pure recombinant Wnt3a protein and their cell behaviors were analyzed including their proliferation, Kit expression, tyrosinase (Tyr) activity, melanin production, dendrite formation and migration. Frizzled-3 was expressed in Tyr-related protein (TRP)-1(+) cells in NC explant cultures, in all 3 melanocyte precursor cell lines and in human melanocytes. Wnt3a increased the population of TRP-1(+) cells, the number of L-3,4-dihydroxyphenylalanine (DOPA)(+) cells and dendrite formation in NC explant cultures. Wnt3a stimulated the proliferation of all 3 melanocyte precursor cell lines in a dose-dependent manner and also stimulated human melanocyte proliferation. Moreover, Wnt3a increased Tyr activity and melanin content of differentiated melanocytes, but did not activate Tyr activity in melanoblasts. Wnt3a stimulated dendrite formation in differentiated melanocytes, but not in melanoblasts. Wnt3a did not affect melanoblast or melanocyte migration. Wnt3a did not induce c-Kit expression in Kit(-) NCCmelb4M5 cells and did not affect c-Kit expression in any cell line tested. Frizzled-3 is constitutively expressed in murine melanocyte precursors, melanocytes and

  2. Homing of hemopoietic precursor cells to the embryonic thymus: characterization of an invasive mechanism induced by chemotactic peptides

    PubMed Central

    1986-01-01

    During embryonic development, T cell precursors migrate to the thymus, where immunocompetency is acquired. Our previous studies have shown that avian hemopoietic precursor cells are recruited to the thymus by chemotactic peptides secreted by thymic epithelial cells (Champion, S., B. A. Imhof, P. Savagner, and J. P. Thiery, 1986, Cell, 44:781-790). In this study, we have characterized the homing of these precursor cells to the thymus in vivo by electron and light microscopy. Hemopoietic precursors could be seen to extravasate from blood or lymphatic vessels, migrate in the mesenchyme, traverse the perithymic basement membrane, and finally intercalate into the thymic epithelium. Labeled hemopoietic precursors injected into the blood circulation also followed the same pathway. Migrating hemopoietic precursor cells were found to express the fibronectin receptor complex. In the presence of thymic chemotactic peptides, hemopoietic precursors traverse a human amniotic basement membrane. This invasive process was inhibited by antibodies to laminin or to fibronectin, two major glycoproteins of the amniotic membrane, by monovalent Fab' fragments of antibodies to the fibronectin receptor, and, finally by synthetic peptides that contain the cell-binding sequence Arg-Gly-Asp-Ser of fibronectin. These results indicate that hemopoietic precursors respond to thymic chemotactic peptides by invasive behavior. Direct interactions between basement membrane components and fibronectin receptors appear to be required for this developmentally regulated invasion process. PMID:3793754

  3. Transplanted miR-219-overexpressing oligodendrocyte precursor cells promoted remyelination and improved functional recovery in a chronic demyelinated model

    PubMed Central

    Fan, Hong-Bin; Chen, Li-Xia; Qu, Xue-Bin; Ren, Chuan-Lu; Wu, Xiu-Xiang; Dong, Fu-Xing; Zhang, Bao-Le; Gao, Dian-Shuai; Yao, Rui-Qin

    2017-01-01

    Oligodendrocyte precursor cells (OPCs) have the ability to repair demyelinated lesions by maturing into myelin-producing oligodendrocytes. Recent evidence suggests that miR-219 helps regulate the differentiation of OPCs into oligodendrocytes. We performed oligodendrocyte differentiation studies using miR-219-overexpressing mouse embryonic stem cells (miR219-mESCs). The self-renewal and multiple differentiation properties of miR219-mESCs were analyzed by the expression of the stage-specific cell markers Nanog, Oct4, nestin, musashi1, GFAP, Tuj1 and O4. MiR-219 accelerated the differentiation of mESC-derived neural precursor cells (NPCs) into OPCs. We further transplanted OPCs derived from miR219-mESCs (miR219-OPCs) into cuprizone-induced chronically demyelinated mice to observe remyelination, which resulted in well-contained oligodendrocyte grafts that migrated along the corpus callosum and matured to express myelin basic protein (MBP). Ultrastructural studies further confirmed the presence of new myelin sheaths. Improved cognitive function in these mice was confirmed by behavioral tests. Importantly, the transplanted miR219-OPCs induced the proliferation of endogenous NPCs. In conclusion, these data demonstrate that miR-219 rapidly transforms mESCs into oligodendrocyte lineage cells and that the transplantation of miR219-OPCs not only promotes remyelination and improves cognitive function but also enhances the proliferation of host endogenous NPCs following chronic demyelination. These results support the potential of a therapeutic role for miR-219 in demyelinating diseases. PMID:28145507

  4. Transplanted miR-219-overexpressing oligodendrocyte precursor cells promoted remyelination and improved functional recovery in a chronic demyelinated model.

    PubMed

    Fan, Hong-Bin; Chen, Li-Xia; Qu, Xue-Bin; Ren, Chuan-Lu; Wu, Xiu-Xiang; Dong, Fu-Xing; Zhang, Bao-Le; Gao, Dian-Shuai; Yao, Rui-Qin

    2017-02-01

    Oligodendrocyte precursor cells (OPCs) have the ability to repair demyelinated lesions by maturing into myelin-producing oligodendrocytes. Recent evidence suggests that miR-219 helps regulate the differentiation of OPCs into oligodendrocytes. We performed oligodendrocyte differentiation studies using miR-219-overexpressing mouse embryonic stem cells (miR219-mESCs). The self-renewal and multiple differentiation properties of miR219-mESCs were analyzed by the expression of the stage-specific cell markers Nanog, Oct4, nestin, musashi1, GFAP, Tuj1 and O4. MiR-219 accelerated the differentiation of mESC-derived neural precursor cells (NPCs) into OPCs. We further transplanted OPCs derived from miR219-mESCs (miR219-OPCs) into cuprizone-induced chronically demyelinated mice to observe remyelination, which resulted in well-contained oligodendrocyte grafts that migrated along the corpus callosum and matured to express myelin basic protein (MBP). Ultrastructural studies further confirmed the presence of new myelin sheaths. Improved cognitive function in these mice was confirmed by behavioral tests. Importantly, the transplanted miR219-OPCs induced the proliferation of endogenous NPCs. In conclusion, these data demonstrate that miR-219 rapidly transforms mESCs into oligodendrocyte lineage cells and that the transplantation of miR219-OPCs not only promotes remyelination and improves cognitive function but also enhances the proliferation of host endogenous NPCs following chronic demyelination. These results support the potential of a therapeutic role for miR-219 in demyelinating diseases.

  5. Preparation of adult muscle fiber-associated stem/precursor cells.

    PubMed

    Conboy, Michael J; Conboy, Irina M

    2010-01-01

    In our studies of muscle regeneration we have developed, modified, and optimized techniques to isolate and study the stem and precursor cells to muscle tissue. Our goals have been to obtain for study muscle fibers in bulk, or the fiber-associated cells, separately from the other cells found in muscle. Using these techniques, myofiber-associated cells may be isolated from neonatal through adult muscle, from resting or from regenerating muscle, thus allowing one to investigate the cellular populations participating during the time course of these events. The protocol is applicable to any age and condition of muscle and may be adapted for other tissues.

  6. (*) Tissue-Specific Extracellular Matrix Enhances Skeletal Muscle Precursor Cell Expansion and Differentiation for Potential Application in Cell Therapy.

    PubMed

    Zhang, Deying; Zhang, Yong; Zhang, Yuanyuan; Yi, Hualin; Wang, Zhan; Wu, Rongpei; He, Dawei; Wei, Guanghui; Wei, Shicheng; Hu, Yun; Deng, Junhong; Criswell, Tracy; Yoo, James; Zhou, Yu; Atala, Anthony

    2017-08-01

    Skeletal muscle precursor cells (MPCs) are considered a key candidate for cell therapy in the treatment of skeletal muscle dysfunction due to injury, disease, or age. However, expansion of a sufficient number of functional skeletal muscle cells in vitro from a small tissue biopsy has been challenging due to changes in phenotypic expression of these cells under traditional culture conditions. Thus, the aim of the study was to develop a better culture system for the expansion and myo-differentiation of MPCs that could further be used for therapy. For this purpose, we developed an ideal method of tissue decellularization and compared the ability of different matrices to support MPC growth and differentiation. Porcine-derived skeletal muscle and liver and kidney extracellular matrix (ECM) were generated by decellularization methods consisting of distilled water, 0.2 mg/mL DNase, or 5% fetal bovine serum. Acellular matrices were further homogenized, dissolved, and combined with a hyaluronic acid-based hydrogel decorated with heparin (ECM-HA-HP). The cell proliferation and myogenic differentiation capacity of human MPCs were assessed when grown on gel alone, ECM, or each ECM-HA-HP substrate. Human MPC proliferation was significantly enhanced when cultured on the ECM-HA-HP substrates compared to the other substrates tested, with the greatest proliferation on the muscle ECM-HA-HP (mECM-HA-HP) substrate. The number of differentiated myotubes was significantly increased on the mECM-HA-HP substrate compared to the other gel-ECM substrates, as well as the numbers of MPCs expressing specific myogenic cell markers (i.e., myosin, desmin, myoD, and myf5). In conclusion, skeletal mECM-HA-HP as a culture substrate provided an optimal culture microenvironment potentially due to its similarity to the in vivo environment. These data suggest a potential use of skeletal muscle-derived ECM gel for the expansion and differentiation of human MPCs for cell-based therapy for skeletal muscle

  7. SVCT2 Is Expressed by Cerebellar Precursor Cells, Which Differentiate into Neurons in Response to Ascorbic Acid.

    PubMed

    Oyarce, Karina; Silva-Alvarez, Carmen; Ferrada, Luciano; Martínez, Fernando; Salazar, Katterine; Nualart, Francisco

    2017-01-17

    Ascorbic acid (AA) is a known antioxidant that participates in a wide range of processes, including stem cell differentiation. It enters the cell through the sodium-ascorbate co-transporter SVCT2, which is mainly expressed by neurons in the adult brain. Here, we have characterized SVCT2 expression in the postnatal cerebellum in situ, a model used for studying neurogenesis, and have identified its expression in granular precursor cells and mature neurons. We have also detected SVCT2 expression in the cerebellar cell line C17.2 and in postnatal cerebellum-derived neurospheres in vitro and have identified a tight relationship between SVCT2 expression and that of the stem cell-like marker nestin. AA supplementation potentiates the neuronal phenotype in cerebellar neural stem cells by increasing the expression of the neuronal marker β III tubulin. Stable over-expression of SVCT2 in C17.2 cells enhances β III tubulin expression, but it also increases cell death, suggesting that AA transporter levels must be finely tuned during neural stem cell differentiation.

  8. Densification Behavior and Performances of C/C Composites Derived from Various Carbon Matrix Precursors

    NASA Astrophysics Data System (ADS)

    Shao, H. C.; Xia, H. Y.; Liu, G. W.; Qiao, G. J.; Xiao, Z. C.; Su, J. M.; Zhang, X. H.; Li, Y. J.

    2014-01-01

    Three types of carbon/carbon (C/C) composites were manufactured by densifying the needled carbon fiber preform through resin and pitch impregnation/carbonization repeatedly, as well as propylene pyrolysis by chemical vapor infiltration plus carbonization after the resin impregnation/carbonization. The densification behavior and performances (involving electric, thermal, and mechanical properties, as well as impurity) of the C/C composites were investigated systematically. The results show that besides the processing and testing conditions, the electric resistivity, thermal conductivity (TC), coefficient of thermal expansion (CTE), strength, and fracture, as well as impurity content and composition of the C/C composites were closely related to the fiber orientation, interfacial bonding between carbon fiber and carbon matrix, material characteristics of the three precursors and the resulting matrix carbons. In particular, the resin-carbon matrix C/C (RC/C) composites had the highest electric resistivity, tensile, and flexural strength, as well as impurity content. Meanwhile, the pitch-carbon matrix C/C (PC/C) composites possessed the highest TC and CTE in the parallel and vertical direction. And most of the performances of pyro-carbon/resin carbon matrix C/C composites were between those of the RC/C and PC/C composites except the impurity content.

  9. Embryonic Nkx2.1-expressing neural precursor cells contribute to the regional heterogeneity of adult V-SVZ neural stem cells.

    PubMed

    Delgado, Ryan N; Lim, Daniel A

    2015-11-15

    The adult ventricular-subventricular zone (V-SVZ) of the lateral ventricle produces several subtypes of olfactory bulb (OB) interneurons throughout life. Neural stem cells (NSCs) within this zone are heterogeneous, with NSCs located in different regions of the lateral ventricle wall generating distinct OB interneuron subtypes. The regional expression of specific transcription factors appears to correspond to such geographical differences in the developmental potential of V-SVZ NSCs. However, the transcriptional definition and developmental origin of V-SVZ NSC regional identity are not well understood. In this study, we found that a population of NSCs in the ventral region of the V-SVZ expresses the transcription factor Nkx2.1 and is derived from Nkx2.1-expressing (Nkx2.1+) embryonic precursors. To follow the fate of Nkx2.1+ cells and their progeny in vivo, we used mice with an Nkx2.1-CreER "knock-in" allele. Nkx2.1+ V-SVZ NSCs labeled in adult mice generated interneurons for the deep granule cell layer of the OB. Embryonic brain Nkx2.1+ precursors labeled at embryonic day 12.5 gave rise to Nkx2.1+ NSCs of the ventral V-SVZ in postnatal and adult mice. Thus, embryonic Nkx2.1+ neural precursors give rise to a population of Nkx2.1+ NSCs in the ventral V-SVZ where they contribute to the regional heterogeneity of V-SVZ NSCs.

  10. Two transcription factors can direct three photoreceptor outcomes from rod precursor cells in mouse retinal development

    PubMed Central

    Ng, Lily; Lu, Ailing; Swaroop, Alok; Sharlin, David; Swaroop, Anand; Forrest, Douglas

    2011-01-01

    The typical mammalian visual system is based upon three photoreceptor types: rods for dim light vision and two types of cones (M and S) for color vision in daylight. However, the process that generates photoreceptor diversity and the cell type in which diversity arises remain unclear. Mice deleted for thyroid hormone receptor ®2 (TR®2) and neural retina leucine zipper factor (NRL) lack M cones and rods, respectively, but gain S cones. We therefore tested the hypothesis that NRL and TR®2 direct a common precursor to a rod, M cone or S cone outcome using Nrlb2/b2 “knock-in” mice that express TR®2 instead of NRL from the endogenous Nrl gene. Nrlb2/b2 mice lacked rods and produced excess M cones in contrast to the excess S cones in Nrl−/− mice. Notably, the presence of both factors yielded rods in Nrl+/b2 mice. The results demonstrate innate plasticity in post-mitotic rod precursors that allows these cells to form three functional photoreceptor types in response to NRL or TRβ2. We also detected precursor cells in normal embryonic retina that transiently co-expressed Nrl and TRβ2, suggesting that some precursors may originate in a plastic state. The plasticity of the precursors revealed in Nrlb2/b2 mice suggests that a two-step transcriptional switch can direct three photoreceptor fates: first, rod versus cone identity dictated by NRL and secondly, if NRL fails to act, M versus S cone identity dictated by TR®2. PMID:21813673

  11. Two transcription factors can direct three photoreceptor outcomes from rod precursor cells in mouse retinal development.

    PubMed

    Ng, Lily; Lu, Ailing; Swaroop, Alok; Sharlin, David S; Swaroop, Anand; Forrest, Douglas

    2011-08-03

    The typical mammalian visual system is based upon three photoreceptor types: rods for dim light vision and two types of cones (M and S) for color vision in daylight. However, the process that generates photoreceptor diversity and the cell type in which diversity arises remain unclear. Mice deleted for thyroid hormone receptor β2 (TRβ2) and neural retina leucine zipper factor (NRL) lack M cones and rods, respectively, but gain S cones. We therefore tested the hypothesis that NRL and TRβ2 direct a common precursor to a rod, M cone, or S cone outcome using Nrl(b2/b2) "knock-in" mice that express TRβ2 instead of NRL from the endogenous Nrl gene. Nrl(b2/b2) mice lacked rods and produced excess M cones in contrast to the excess S cones in Nrl(-/-) mice. Notably, the presence of both factors yielded rods in Nrl(+/b2) mice. The results demonstrate innate plasticity in postmitotic rod precursors that allows these cells to form three functional photoreceptor types in response to NRL or TRβ2. We also detected precursor cells in normal embryonic retina that transiently coexpressed Nrl and TRβ2, suggesting that some precursors may originate in a plastic state. The plasticity of the precursors revealed in Nrl(b2/b2) mice suggests that a two-step transcriptional switch can direct three photoreceptor fates: first, rod versus cone identity dictated by NRL, and second, if NRL fails to act, M versus S cone identity dictated by TRβ2.

  12. BMP4 can generate primordial germ cells from bone-marrow-derived pluripotent stem cells.

    PubMed

    Shirazi, Reza; Zarnani, Amir Hassan; Soleimani, Masoud; Abdolvahabi, Mir Abbas; Nayernia, Karim; Ragerdi Kashani, Iraj

    2012-01-01

    Evidence of germ cell derivation from embryonic and somatic stem cells provides an in vitro model for the study of germ cell development, associated epigenetic modification and mammalian gametogenesis. More importantly, in vitro derived gametes also represent a potential strategy for treating infertility. In mammals, male and female gametes, oocyte and sperm, are derived from a specific cell population, PGCs (primordial germ cells) that segregate early in embryogenesis. We have isolated pluripotent SSEA-1+ (stage-specific embryonic antigen-1+) cells from mice bone marrow using a MACS (magnetic-activated cell sorting) system. SSEA-1+ cells were directly separated from the suspension of MMCs (murine mononuclear cells) harvested from bone marrow of 2-4-week-old mice. Flow-cytometry assay immediately after sorting and culturing under undifferentiated condition showed 55±7% and 87±4% purity respectively. RT-PCR (reverse transcription-PCR) analysis after differentiation of SSEA-1+ cells into derivations of three germ layers showed the pluripotency properties of isolated cells. SSEA-1+ cells were induced to differentiate along germ cell lineage by adding BMP4 (bone morphogenic factor-4) to the medium. Regarding the expression of germ cell markers (PGCs, male and female germ cell lineage), it was found that adding exogenous BMP4 to culture medium could differentiate pluripotent SSEA-1+ cells isolated from an adult tissue into gamete precursors, PGCs. Differentiated cells expressed specific molecular markers of PGCs, including Oct4, fragilis, Stella and Mvh (mouse vasa homologue). Therefore BMP4 is insufficient to induce SSEA-1+ cells derived from PGCs to develop further into late germ cells in vitro.

  13. Derivation and characterization of a pig embryonic stem cell-derived exocrine pancreatic cell line

    USDA-ARS?s Scientific Manuscript database

    The establishment and initial characterization of a pig embryonic stem cell-derived pancreatic cell line, PICM-31, and a colony-cloned derivative cell line, PICM-31A, is described. The cell lines were propagated for several months at split ratios of 1:3 or 1:5 at each passage on STO feeder cells af...

  14. Phenotypic Screening Identifies Modulators of Amyloid Precursor Protein Processing in Human Stem Cell Models of Alzheimer's Disease.

    PubMed

    Brownjohn, Philip W; Smith, James; Portelius, Erik; Serneels, Lutgarde; Kvartsberg, Hlin; De Strooper, Bart; Blennow, Kaj; Zetterberg, Henrik; Livesey, Frederick J

    2017-03-06

    Human stem cell models have the potential to provide platforms for phenotypic screens to identify candidate treatments and cellular pathways involved in the pathogenesis of neurodegenerative disorders. Amyloid precursor protein (APP) processing and the accumulation of APP-derived amyloid β (Aβ) peptides are key processes in Alzheimer's disease (AD). We designed a phenotypic small-molecule screen to identify modulators of APP processing in trisomy 21/Down syndrome neurons, a complex genetic model of AD. We identified the avermectins, commonly used as anthelmintics, as compounds that increase the relative production of short Aβ peptides at the expense of longer, potentially more toxic peptides. Further studies demonstrated that this effect is not due to an interaction with the core γ-secretase responsible for Aβ production. This study demonstrates the feasibility of phenotypic drug screening in human stem cell models of Alzheimer-type dementia, and points to possibilities for indirectly modulating APP processing, independently of γ-secretase modulation.

  15. N-Acetyl-L-Cystein downregulates beta-amyloid precursor protein gene transcription in human neuroblastoma cells.

    PubMed

    Studer, R; Baysang, G; Brack, C

    2001-01-01

    The causes for the sporadic form of Alzheimer's disease (AD) are still poorly understood, except from the fact that age is an important risk factor. The main component of the characteristic amyloid plaques in brains of AD patients are Abeta peptides, derivatives of the amyloid precursor protein APP. Oxidative stress may contribute to the aetiology of AD by dysregulation of APP metabolism. Overexpression of the APP gene could result in an increased secretion of neurotoxic Abeta peptides, while preventing the overexpression might be protective. We here report that the antioxidant N-Acetyl-L-Cystein (NAC) downregulates APP gene transcription in human neuroblastoma cells. The effect is reversible when cells are returned to NAC free medium. These results open up new possibilities for the development of therapeutic agents that intervene at the transcriptional level.

  16. Brain-derived neurotrophic factor (BDNF) and its precursor (proBDNF) in genetically defined fear-induced aggression.

    PubMed

    Ilchibaeva, Tatiana V; Kondaurova, Elena M; Tsybko, Anton S; Kozhemyakina, Rimma V; Popova, Nina K; Naumenko, Vladimir S

    2015-09-01

    The brain-derived neurotrophic factor (BDNF), its precursor (proBDNF) and BDNF mRNA levels were studied in the brain of wild rats selectively bred for more than 70 generations for either high level or for the lack of affective aggressiveness towards man. Significant increase of BDNF mRNA level in the frontal cortex and increase of BDNF level in the hippocampus of aggressive rats was revealed. In the midbrain and hippocampus of aggressive rats proBDNF level was increased, whereas BDNF/proBDNF ratio was reduced suggesting the prevalence and increased influence of proBDNF in highly aggressive rats. In the frontal cortex, proBDNF level in aggressive rats was decreased. Thus, considerable structure-specific differences in BDNF and proBDNF levels as well as in BDNF gene expression between highly aggressive and nonaggressive rats were shown. The data suggested the implication of BDNF and its precursor proBDNF in the mechanism of aggressiveness and in the creation of either aggressive or nonaggressive phenotype.

  17. Polysiloxanes derived from the controlled hydrolysis of tetraethoxysilane as precursors to silica for use in ceramic processing

    NASA Technical Reports Server (NTRS)

    Philipp, Warren H.

    1990-01-01

    Synthesis, properties, and potential applications in ceramic processing for two polysiloxane silica precursors derived from the controlled hydrolysis of tetraethoxysilane (TEOS) are presented. The higher molecular weight TEOS-A is a thick adhesive liquid of viscosity 8000 to 12,000 c.p. having a SiO2 char yield of about 55 percent. The lower molecular weight TEOS-B is a more fluid liquid of viscosity 150 to 200 c.p. having a SiO2 char yield of about 52 percent. The acid catalyzed hydrolysis of TEOS to hydrated silica gel goes through a series of polysiloxane intermediates. The rate of this transition increases with the quantity of water added to the TEOS; thus, for ease of polymer isolation, the amount of water added must be carefully determined so as to produce the desired polymer in a reasonable time. The water to TEOS mole ratio falls in the narrow range of 1.05 for TEOS-A and 0.99 for TEOS-B. Further polymerization or gelation is prevented by storing at -5 C in a freezer. Both polysiloxanes thermoset to a glassy solid at 115 C. The liquid polymers are organic in nature in that they are miscible with toluene and ethanol, slightly souble in heptane, but immiscible with water. For both polymers, results on viscosity versus time are given at several temperatures and water additions. Based on these results, some examples of practical utilization of the precursors for ceramic fabrication are given.

  18. Differentiation of retinal ganglion cells and photoreceptor precursors from mouse induced pluripotent stem cells carrying an Atoh7/Math5 lineage reporter.

    PubMed

    Xie, Bin-Bin; Zhang, Xiang-Mei; Hashimoto, Takao; Tien, Amy H; Chen, Andrew; Ge, Jian; Yang, Xian-Jie

    2014-01-01

    The neural retina is a critical component of the visual system, which provides the majority of sensory input in humans. Various retinal degenerative diseases can result in the permanent loss of retinal neurons, especially the light-sensing photoreceptors and the centrally projecting retinal ganglion cells (RGCs). The replenishment of lost RGCs and the repair of optic nerve damage are particularly challenging, as both RGC specification and their subsequent axonal growth and projection involve complex and precise regulation. To explore the developmental potential of pluripotent stem cell-derived neural progenitors, we have established mouse iPS cells that allow cell lineage tracing of progenitors that have expressed Atoh7/Math5, a bHLH transcription factor required for RGC production. These Atoh7 lineage reporter iPS cells encode Cre to replace one copy of the endogenous Atoh7 gene and a Cre-dependent YFP reporter in the ROSA locus. In addition, they express pluripotent markers and are capable of generating teratomas in vivo. Under anterior neural induction and neurogenic conditions in vitro, the Atoh7-Cre/ROSA-YFP iPS cells differentiate into neurons that co-express various RGC markers and YFP, indicating that these neurons are derived from Atoh7-expressing progenitors. Consistent with previous in vivo cell lineage studies, the Atoh7-Cre/ROSA-YFP iPS cells also give rise to a subset of Crx-positive photoreceptor precursors. Furthermore, inhibition of Notch signaling in the iPSC cultures results in a significant increase of YFP-positive RGCs and photoreceptor precursors. Together, these results show that Atoh7-Cre/ROSA-YFP iPS cells can be used to monitor the development and survival of RGCs and photoreceptors from pluripotent stem cells.

  19. Comparison of melatonin with growth factors in promoting precursor cells proliferation in adult mouse subventricular zone

    PubMed Central

    Sotthibundhu, Areechun; Ekthuwapranee, Kasima; Govitrapong, Piyarat

    2016-01-01

    Melatonin, secreted mainly by the pineal gland, plays roles in various physiological functions including protecting cell death. We showed in previous study that the proliferation and differentiation of precursor cells from the adult mouse subventricular zone (SVZ) can be modulated by melatonin via the MT1 melatonin receptor. Since melatonin and epidermal growth factor receptor (EGFR) share some signaling pathway components, we investigated whether melatonin can promote the proliferation of precursor cells from the adult mouse SVZ via the extracellular signal-regulated protein kinase /mitogen-activated protein kinase (ERK/MAPK) pathways in comparison with epidermal growth factor (EGF). Melatonin-induced ERK/MAPK pathways compared with EGF were measured by using in vitro and vivo models. We used neurosphere proliferation assay, immunocytochemistry, and immuno-blotting to analyze significant differences between melatonin and growth factor treatment. We also used specific antagonist and inhibitors to confirm the exactly signaling pathway including luzindole and U0126. We found that significant increase in proliferation was observed when two growth factors (EGF+bFGF) and melatonin were used simultaneously compared with EGF + bFGF or compared with melatonin alone. In addition, the present result suggested the synergistic effect occurred of melatonin and growth factors on the activating the ERK/MAPK pathway. This study exhibited that melatonin could act as a trophic factor, increasing proliferation in precursor cells mediated through the melatonin receptor coupled to ERK/MAPK signaling pathways. Understanding the mechanism by which melatonin regulates precursor cells may conduct to the development of novel strategies for neurodegenerative disease therapy. PMID:28275319

  20. Extensive Ex Vivo Expansion of Functional Human Erythroid Precursors Established From Umbilical Cord Blood Cells by Defined Factors

    PubMed Central

    Huang, Xiaosong; Shah, Siddharth; Wang, Jing; Ye, Zhaohui; Dowey, Sarah N; Tsang, Kit Man; Mendelsohn, Laurel G; Kato, Gregory J; Kickler, Thomas S; Cheng, Linzhao

    2014-01-01

    There is a constant shortage of red blood cells (RBCs) from sufficiently matched donors for patients who need chronic transfusion. Ex vivo expansion and maturation of human erythroid precursors (erythroblasts) from the patients or optimally matched donors could represent a potential solution. Proliferating erythroblasts can be expanded from umbilical cord blood mononuclear cells (CB MNCs) ex vivo for 106–107-fold (in ~50 days) before proliferation arrest and reaching sufficient number for broad application. Here, we report that ectopic expression of three genetic factors (Sox2, c-Myc, and an shRNA against TP53 gene) associated with iPSC derivation enables CB-derived erythroblasts to undergo extended expansion (~1068-fold in ~12 months) in a serum-free culture condition without change of cell identity or function. These expanding erythroblasts maintain immature erythroblast phenotypes and morphology, a normal diploid karyotype and dependence on a specific combination of growth factors for proliferation throughout expansion period. When being switched to a terminal differentiation condition, these immortalized erythroblasts gradually exit cell cycle, decrease cell size, accumulate hemoglobin, condense nuclei and eventually give rise to enucleated hemoglobin-containing erythrocytes that can bind and release oxygen. Our result may ultimately lead to an alternative approach to generate unlimited numbers of RBCs for personalized transfusion medicine. PMID:24002691

  1. In vitro effect of glutathione precursors on cytotoxicity of amino acids to human mesothelial cells.

    PubMed

    Grzybowski, A E

    1999-09-01

    Amino acids (AA) which were proposed as an alternative osmotically active agents in dialysates are toxic to human peritoneal mesothelial cells (HPMC) due to disturbance of the antioxidant-oxidant balance in cells by reducing level of glutathione. We assessed if the addition intracellular glutathione precursors: N-acetyl-cysteine (NAC), tioproline (TP), L--2-oxo--4-thiazolidine acid (PC), and glutathione (GSH) could reduce the cytotoxicity of AA, as measured by inhibition of cells proliferation and disorders of intracellular 86Rb transport. HPMC were obtained from omentum from nonuremic donors and cultured in in vitro conditions. The HPMC proliferation capacity was assessed indirectly by the 3H-methyl-thymidine incorporation assay. The injury to HPMC membrane integrity was assessed by the release of radioisotope molecules of 86Rb from the prelabelled cells. We have found that AA diminished the intracellular potassium (86Rb) influx. Supplementation of AA mixture with NAC enhanced the total 86Rb influx into HMC. Other precursors of intracellular glutathione (TP,PC,GSH) tested in the presence of AA significantly stimulated intracellular transport of 86Rb via Na,K-ATPase dependent channel, but the total intracellular transport of 86Rb was still lower than in control. HMC proliferation was significantly inhibited by AA what was measured by incorporation of H-metyl-tymidine. In the presence of NAC inhibition of HMC proliferation caused by AA was weaker. Our results suggest that some of intracellular glutathione precursors may reduce the disturbances of the HMC function caused by AA.

  2. Lineage-Specific Genes Are Prominent DNA Damage Hotspots during Leukemic Transformation of B Cell Precursors.

    PubMed

    Boulianne, Bryant; Robinson, Mark E; May, Philippa C; Castellano, Leandro; Blighe, Kevin; Thomas, Jennifer; Reid, Alistair; Müschen, Markus; Apperley, Jane F; Stebbing, Justin; Feldhahn, Niklas

    2017-02-14

    In human leukemia, lineage-specific genes represent predominant targets of deletion, with lymphoid-specific genes frequently affected in lymphoid leukemia and myeloid-specific genes in myeloid leukemia. To investigate the basis of lineage-specific alterations, we analyzed global DNA damage in primary B cell precursors expressing leukemia-inducing oncogenes by ChIP-seq. We identified more than 1,000 sensitive regions, of which B lineage-specific genes constitute the most prominent targets. Identified hotspots at B lineage genes relate to DNA-DSBs, affect genes that harbor genomic lesions in human leukemia, and associate with ectopic deletion in successfully transformed cells. Furthermore, we show that most identified regions overlap with gene bodies of highly expressed genes and that induction of a myeloid lineage phenotype in transformed B cell precursors promotes de novo DNA damage at myeloid loci. Hence, we demonstrate that lineage-specific transcription predisposes lineage-specific genes in transformed B cell precursors to DNA damage, which is likely to promote the frequent alteration of lineage-specific genes in human leukemia. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  3. Subcellular sites of processing of precursors to neurosecretory peptides in the bag cells of Aplysia: inferences from the effects of monensin, FCCP, and chloroquine.

    PubMed

    Yates, M E; Berry, R W

    1984-03-01

    The effects of the sodium ionophore monensin were examined in the bag cells of Aplysia californica in order to identify the subcellular sites of processing of precursors to their neurosecretory products. Incubation of bag cells in media containing 10 microM monensin led to a marked disruption of the morphology of the Golgi apparatus without affecting that of other organelles. Exposure of bag cells to monensin led to a significant impairment of processing of the largest precursor and of an intermediate protein which gives rise to the immediate precursors to the final secreted products, the egg-laying hormone (ELH) and the acidic peptide (AP). Furthermore, ELH and AP were never produced in the presence of monensin during the time course of these experiments. When axonal transport was allowed to proceed, the contents of bag-cell terminals indicated that the intermediate protein is the first to be packaged in Golgi-derived vesicles, and in monensin-treated cells may be transported without being processed further. In contrast to these results, the protonophore FCCP-impaired precursor and intermediate cleavage equally, indicating that monensin and FCCP have different effects on intracellular transport and precursor processing. These data are interpreted to indicate that the largest ELH-AP precursor is normally processed within the Golgi apparatus, and that the disruption of this organelle induced by monensin produces the impairment seen in its processing. The impairment of cleavage of the intermediate species, and the blockade of production of AP and ELH, are probably the result of monensin-induced impairment of production of proteolytically competent secretory granules by the Golgi apparatus.

  4. Adult neurogenesis and repair of the adult CNS with neural progenitors, precursors, and stem cells.

    PubMed

    Emsley, Jason G; Mitchell, Bartley D; Kempermann, Gerd; Macklis, Jeffrey D

    2005-04-01

    Recent work in neuroscience has shown that the adult central nervous system contains neural progenitors, precursors, and stem cells that are capable of generating new neurons, astrocytes, and oligodendrocytes. While challenging previous dogma that no new neurons are born in the adult mammalian CNS, these findings bring with them future possibilities for the development of novel neural repair strategies. The purpose of this review is to present current knowledge about constitutively occurring adult mammalian neurogenesis, to highlight the critical differences between "neurogenic" and "non-neurogenic" regions in the adult brain, and to describe the cardinal features of two well-described neurogenic regions-the subventricular zone/olfactory bulb system, and the dentate gyrus of the hippocampus. We also provide an overview of currently used models for studying neural precursors in vitro, mention some precursor transplantation models, and emphasize that, in this rapidly growing field of neuroscience, one must take caution with respect to a variety of methodological considerations for studying neural precursor cells both in vitro and in vivo. The possibility of repairing neural circuitry by manipulating neurogenesis is an intriguing one, and, therefore, we also review recent efforts to understand the conditions under which neurogenesis can be induced in non-neurogenic regions of the adult CNS. This work aims toward molecular and cellular manipulation of endogenous neural precursors in situ, without transplantation. We conclude this review with a discussion of what the function might be of newly generated neurons in the adult brain and provide a summary of current thinking about the consequences of disturbed adult neurogenesis and the reaction of neurogenic regions to disease.

  5. Adult neurogenesis and cellular brain repair with neural progenitors, precursors and stem cells.

    PubMed

    Sohur, U Shivraj; Emsley, Jason G; Mitchell, Bartley D; Macklis, Jeffrey D

    2006-09-29

    Recent work in neuroscience has shown that the adult central nervous system (CNS) contains neural progenitors, precursors and stem cells that are capable of generating new neurons, astrocytes and oligodendrocytes. While challenging the previous dogma that no new neurons are born in the adult mammalian CNS, these findings bring with them the future possibilities for development of novel neural repair strategies. The purpose of this review is to present the current knowledge about constitutively occurring adult mammalian neurogenesis, highlight the critical differences between 'neurogenic' and 'non-neurogenic' regions in the adult brain, and describe the cardinal features of two well-described neurogenic regions-the subventricular zone/olfactory bulb system and the dentate gyrus of the hippocampus. We also provide an overview of presently used models for studying neural precursors in vitro, mention some precursor transplantation models and emphasize that, in this rapidly growing field of neuroscience, one must be cautious with respect to a variety of methodological considerations for studying neural precursor cells both in vitro and in vivo. The possibility of repairing neural circuitry by manipulating neurogenesis is an intriguing one, and, therefore, we also review recent efforts to understand the conditions under which neurogenesis can be induced in non-neurogenic regions of the adult CNS. This work aims towards molecular and cellular manipulation of endogenous neural precursors in situ, without transplantation. We conclude this review with a discussion of what might be the function of newly generated neurons in the adult brain, and provide a summary of present thinking about the consequences of disturbed adult neurogenesis and the reaction of neurogenic regions to disease.

  6. Adult neurogenesis and cellular brain repair with neural progenitors, precursors and stem cells

    PubMed Central

    Shivraj Sohur, U; Emsley, Jason G; Mitchell, Bartley D; Macklis, Jeffrey D

    2006-01-01

    Recent work in neuroscience has shown that the adult central nervous system (CNS) contains neural progenitors, precursors and stem cells that are capable of generating new neurons, astrocytes and oligodendrocytes. While challenging the previous dogma that no new neurons are born in the adult mammalian CNS, these findings bring with them the future possibilities for development of novel neural repair strategies. The purpose of this review is to present the current knowledge about constitutively occurring adult mammalian neurogenesis, highlight the critical differences between ‘neurogenic’ and ‘non-neurogenic’ regions in the adult brain, and describe the cardinal features of two well-described neurogenic regions—the subventricular zone/olfactory bulb system and the dentate gyrus of the hippocampus. We also provide an overview of presently used models for studying neural precursors in vitro, mention some precursor transplantation models and emphasize that, in this rapidly growing field of neuroscience, one must be cautious with respect to a variety of methodological considerations for studying neural precursor cells both in vitro and in vivo. The possibility of repairing neural circuitry by manipulating neurogenesis is an intriguing one, and, therefore, we also review recent efforts to understand the conditions under which neurogenesis can be induced in non-neurogenic regions of the adult CNS. This work aims towards molecular and cellular manipulation of endogenous neural precursors in situ, without transplantation. We conclude this review with a discussion of what might be the function of newly generated neurons in the adult brain, and provide a summary of present thinking about the consequences of disturbed adult neurogenesis and the reaction of neurogenic regions to disease. PMID:16939970

  7. Late outgrowth endothelial cells resemble mature endothelial cells and are not derived from bone marrow.

    PubMed

    Tura, Olga; Skinner, Elizabeth M; Barclay, G Robin; Samuel, Kay; Gallagher, Ronald C J; Brittan, Mairi; Hadoke, Patrick W F; Newby, David E; Turner, Marc L; Mills, Nicholas L

    2013-02-01

    A decade of research has sought to identify circulating endothelial progenitor cells (EPC) in order to harness their potential for cardiovascular regeneration. Endothelial outgrowth cells (EOC) most closely fulfil the criteria for an EPC, but their origin remains obscure. Our aim was to identify the source and precursor of EOC and to assess their regenerative potential compared to mature endothelial cells. EOC are readily isolated from umbilical cord blood (6/6 donors) and peripheral blood mononuclear cells (4/6 donors) but not from bone marrow (0/6) or peripheral blood following mobilization with granulocyte-colony stimulating factor (0/6 donors). Enrichment and depletion of blood mononuclear cells demonstrated that EOC are confined to the CD34(+)CD133(-)CD146(+) cell fraction. EOC derived from blood mononuclear cells are indistinguishable from mature human umbilical vein endothelial cells (HUVEC) by morphology, surface antigen expression, immunohistochemistry, real-time polymerase chain reaction, proliferation, and functional assessments. In a subcutaneous sponge model of angiogenesis, both EOC and HUVEC contribute to de novo blood vessel formation giving rise to a similar number of vessels (7.0 ± 2.7 vs. 6.6 ± 3.7 vessels, respectively, n = 9). Bone marrow-derived outgrowth cells isolated under the same conditions expressed mesenchymal markers rather than endothelial cell markers and did not contribute to blood vessels in vivo. In this article, we confirm that EOC arise from CD34(+)CD133(-)CD146(+) mononuclear cells and are similar, if not identical, to mature endothelial cells. Our findings suggest that EOC do not arise from bone marrow and challenge the concept of a bone marrow-derived circulating precursor for endothelial cells. Copyright © 2012 AlphaMed Press.

  8. Emerging roles for the amyloid precursor protein and derived peptides in the regulation of cellular and systemic metabolism.

    PubMed

    Czeczor, Juliane K; McGee, Sean L

    2017-03-28

    The amyloid precursor protein (APP) is a transmembrane protein that can be cleaved by proteases through two different pathways to yield a number of small peptides, each with distinct physiological properties and functions. It has been extensively studied in the context of Alzheimer's disease, with the APP-derived amyloid beta (Aβ) peptide being a major constituent of the amyloid plaques observed in this disease. It has been known for some time that APP can regulate neuronal metabolism, however this review will examine evidence that APP and its peptides can also regulate key metabolic processes such as insulin action, lipid synthesis and storage and mitochondrial function in peripheral tissues. This review will present a hypothesis that amyloidogenic processing of APP in peripheral tissues plays a key role in the response to nutrient excess and that this could contribute to the pathogenesis of metabolic diseases such as obesity and type 2 diabetes (T2D). This article is protected by copyright. All rights reserved.

  9. In Senescence, Age-associated B Cells (ABC) Secrete TNFα and Inhibit Survival of B Cell Precursors1

    PubMed Central

    Ratliff, Michelle; Alter, Sarah; Frasca, Daniela; Blomberg, Bonnie B.; Riley, Richard L.

    2013-01-01

    Aged mice exhibit ~ 5-10 fold increases in an ordinarily minor CD21/35− CD23− mature B cell subset termed age-associated B cells (ABC). ABC from old, but not young, mice induce apoptosis in pro-B cells directly through secretion of TNFα. In addition, aged ABC, via TNFα, stimulate bone marrow cells to suppress pro-B cell growth. ABC effects can be prevented by the anti-inflammatory cytokine IL-10. Notably, CD21/35+ CD23+ follicular (FO) splenic and FO-like recirculating bone marrow B cells in both young and aged mice contain a subpopulation which produces IL-10. Unlike young adult FO B cells, old FO B cells also produce TNFα; however, secretion of IL-10 within this B cell population ameliorates the TNFα-mediated effects on B cell precursors. Loss of B cell precursors in the bone marrow of old mice in vivo was significantly associated with increased ABC relative to recirculating FO-like B cells. Adoptive transfer of aged ABC into RAG-2 KO recipients resulted in significant losses of pro-B cells within the bone marrow. These results suggest that alterations in B cell composition during old age, in particular the increase in ABC within the B cell compartments contribute to a pro-inflammatory environment within the bone marrow. This provides a mechanism of inappropriate B cell “feedback” which promotes down-regulation of B lymphopoiesis in old age. PMID:23410004

  10. Ankfy1 is dispensable for neural stem/precursor cell development.

    PubMed

    Weng, Chao; Ding, Man; Chang, Lian-Sheng; Ren, Ming-Xin; Zhang, Hong-Feng; Lu, Zu-Neng; Fu, Hui

    2016-11-01

    There are few studies on the membrane protein Ankfy1. We have found Ankfy1 is specifically expressed in neural stem/precursor cells during early development in mice (murine). To further explore Ankfy1 function in neural development, we developed a gene knockout mouse with a mixed Balb/C and C57/BL6 genetic background. Using immunofluorescence and in situ hybridization, neural defects were absent in mixed genetic Ankfy1 null mice during development and in adults up to 2 months old. However, Ankfy1 gene knockout mice with a pure genetic background were found to be lethal in the C57/BL6 inbred mice embryos, even after seven generations of backcrossing. Polymerase chain reaction confirmed homozygotes were unattainable as early as embryonic day 11.5. We conclude that Ankfy1 protein is dispensable in neural stem/precursor cells, but could be critical for early embryonic murine development, depending on the genetic background.

  11. Circulating myeloid and lymphoid precursor dendritic cells are clonally involved in myelodysplastic syndromes.

    PubMed

    Ma, L; Delforge, M; van Duppen, V; Verhoef, G; Emanuel, B; Boogaerts, M; Hagemeijer, A; Vandenberghe, P

    2004-09-01

    Circulating myeloid and lymphoid precursor dendritic cell (pDC) counts were determined in peripheral blood from 22 patients with myelodysplastic syndromes (MDS) by a single-platform flow cytometric protocol. The absolute count of myeloid and lymphoid pDC, as well as their relative number (as proportion of mononuclear cells or total leukocytes) was significantly lower in MDS (n=22) than in healthy controls (n=41). In 11 patients with chromosomal aberrations, purified pDC were examined by interphase fluorescence in situ hybridization. This revealed clonal involvement of myeloid as well as lymphoid pDC in all of them. These data therefore strongly suggest that myeloid and lymphoid pDC share a common precursor. Whether reduced peripheral blood counts of pDC contribute to the immunological abnormalities observed in MDS remains to be investigated.

  12. Identification of myocardial and vascular precursor cells in human and mouse epicardium.

    PubMed

    Limana, Federica; Zacheo, Antonella; Mocini, David; Mangoni, Antonella; Borsellino, Giovanna; Diamantini, Adamo; De Mori, Roberta; Battistini, Luca; Vigna, Elisa; Santini, Massimo; Loiaconi, Vincenzo; Pompilio, Giulio; Germani, Antonia; Capogrossi, Maurizio C

    2007-12-07

    During cardiac development, the epicardium is the source of multipotent mesenchymal cells, which give rise to endothelial and smooth muscle cells in coronary vessels and also, possibly, to cardiomyocytes. The aim of the present study was to determine whether stem cells are retained in the adult human and murine epicardium and to investigate the regenerative potential of these cells following acute myocardial infarction. We show that c-kit(+) and CD34(+) cells can indeed be detected in human fetal and adult epicardium and that they represent 2 distinct populations. Both subsets of cells were negative for CD45, a cell surface marker that identifies the hematopoietic cell lineage. Immunofluorescence revealed that freshly isolated c-kit(+) and CD34(+) cells expressed early and late cardiac transcription factors and could acquire an endothelial phenotype in vitro. In the murine model of myocardial infarction, there was an increase in the absolute number and proliferation of epicardial c-kit(+) cells 3 days after coronary ligation; at this time point, epicardial c-kit(+) cells were identified in the subepicardial space and expressed GATA4. Furthermore, 1 week after myocardial infarction, cells coexpressing c-kit(+), together with endothelial or smooth muscle cell markers, were identified in the wall of subepicardial blood vessels. In summary, the postnatal epicardium contains a cell population with stem cell characteristics that retains the ability to give rise to myocardial precursors and vascular cells. These cells may play a role in the regenerative response to cardiac damage.

  13. CD22 is required for formation of memory B cell precursors within germinal centers

    PubMed Central

    Chappell, Craig P.; Draves, Kevin E.

    2017-01-01

    CD22 is a BCR co-receptor that regulates B cell signaling, proliferation and survival and is required for T cell-independent Ab responses. To investigate the role of CD22 during T cell-dependent (TD) Ab responses and memory B cell formation, we analyzed Ag-specific B cell responses generated by wild-type (WT) or CD22-/- B cells following immunization with a TD Ag. CD22-/- B cells mounted normal early Ab responses yet failed to generate either memory B cells or long-lived plasma cells, whereas WT B cells formed both populations. Surprisingly, B cell expansion and germinal center (GC) differentiation were comparable between WT and CD22-/- B cells. CD22-/- B cells, however, were significantly less capable of generating a population of CXCR4hiCD38hi GC B cells, which we propose represent memory B cell precursors within GCs. These results demonstrate a novel role for CD22 during TD humoral responses evident during primary GC formation and underscore that CD22 functions not only during B cell maturation but also during responses to both TD and T cell-independent antigens. PMID:28346517

  14. Frizzled signalling controls orientation of asymmetric sense organ precursor cell divisions in Drosophila.

    PubMed

    Gho, M; Schweisguth, F

    1998-05-14

    During metazoan development, cell-fate diversity is brought about, in part, by asymmetric cell divisions. In Drosophila, bristle mechanosensory organs are composed of four different cells that originate from a single precursor cell, pI, after two rounds of asymmetric division. At each division, distinct fates are conferred on sister cells by the asymmetric segregation of Numb, a negative regulator of Notch signalling. Here we show that the orientation of the mitotic spindles and the localization of the Numb crescent follow a stereotyped pattern. Mitosis of pI is orientated parallel to the anteroposterior axis of the fly. We show that signalling mediated by the Frizzled receptor polarizes pI along this axis, thereby specifying the orientation of the mitotic spindle and positioning the Numb crescent. The mitoses of the two cells produced by mitosis of pI are orientated parallel and orthogonal, respectively, to the division axis of pI. This difference in cell-division orientation is largely independent of the identity of the secondary precursor cells, and is regulated by Frizzled-independent mechanisms.

  15. Ionotropic glutamate receptor antagonists inhibit the proliferation of granule cell precursors in the adult brain after seizures induced by pentylenetrazol.

    PubMed

    Jiang, Wen; Wolfe, Ken; Xiao, Lan; Zhang, Zhi-Jun; Huang, Yuan-Gui; Zhang, Xia

    2004-09-10

    Seizures have been shown to promote the proliferation of granule cell precursors in the adult brain, but the underlying mechanisms remain largely unknown. Using systemic bromodeoxyuridine (BrdU) to label dividing cells, we examined the effects of selective ionotropic glutamate receptor antagonists on granule cell precursor proliferation in adult rats after pentylenetrazol (PTZ)-induced generalized clonic seizures. We found that the NMDA receptor antagonist MK-801 significantly inhibited behavioral and EEG seizures and completely blocked seizure-induced increase in the number of BrdU-labeled cells in the dentate gyrus. Although the AMPA/KA receptor antagonist DNQX was not observed to affect seizures, it significantly suppressed the number of BrdU-labeled cells in the dentate gyrus. Double immunohistochemical staining showed that both the mature granule cells and the majority of BrdU-labeled, mitotically active cells expressed the NMDA receptor subunit NR1 and the AMPA/KA receptor subunit GluR2. Because accumulated evidence showed that mild seizures are sufficient to promote precursor cell proliferation, the present findings that MK-801 inhibited seizures and completely blocked seizure-induced increase in precursor cell proliferation suggest that the direct blockade action of MK-801 on NMDA receptors on the granule cell precursors may play an important role in blocking seizure-induced precursor cell proliferation. The suppression of seizure-induced proliferation of granule cell precursors by DNQX may be achieved by the direct action of DNQX on AMPA/KA receptors on the granule cell precursors. Thus, our findings indicate that seizures may promote cell proliferation in the adult rat dentate gyrus through glutamatergic mechanisms acting on both NMDA and AMPA/KA receptors.

  16. Sсandium(III) Beta-diketonate Derivatives as Precursors for Oxide Film Deposition by CVD

    NASA Astrophysics Data System (ADS)

    Zherikova, Kseniya V.; Zelenina, Ludmila N.; Chusova, Tamara P.; Morozova, Natalia B.; Trubin, Sergey V.; Vikulova, Eugeniia S.

    Complexes with acetylacetone Sc(acac)3, dipivaloylmethane Sc(thd)3, 2,2,6,6-tetramethyl-4-fluoro-3,5-heptanedione Sc(tfhd)3, pivaloyltrifluoroacetone Sc(ptac)3, trifluoroacetylacetone Sc(tfac)3, and hexafluoroacetylacetone Sc(hfac)3 were synthesized, purified and identified by elemental analysis, m.p., IR and NMR spectroscopy, and mass spectrometry. The thermal behaviour of the synthesized compounds in the solid state was investigated by the method of difference-scanning calorimetry in vacuum. As a result the thermodynamic characteristics of the melting processes were determined. The temperature dependences of saturated and unsaturated vapour pressure of complexes under study were measured by static method with membrane-gauge manometers. The average molecular weight of gas calculated from the experimental data on unsaturated vapours using ideal gas law was close to the molecular weight of monomer for all investigated compounds. Decomposition temperatures of compounds under study were defined as the temperature above that pressure changes became irreversible. The information about melting and decomposition processes were taken into account at measuring saturated vapour pressure of complexes. The row of volatility Sc(hfac)3 > Sc(ptac)3 > Sc(tfac)3 > Sc(thd)3 ≥ Sc(tfhd)3 > Sc(acac)3 was determined from the p-T dependences obtained. Above information about thermal behaviour of complexes enabled one to use it as a guide for CVD experiments aimed at achieving oxide films with high optical properties. Precursor chosen for film deposition was Sc(thd)3 The Sc2O3 film deposition conditions were following: the gas-carrier rate 1 l/h, He gas-reagent rate 10 l/h, total pressure 10 Torr, evaporator temperature 105-110̊С, substrate temperature 450- 650̊С, substrate Si(100). Ellipsometry was applied to characterize the film thickness and refractive index. The morphology and the composition of the films were determined with XPS and SEM.

  17. New melanogenesis and photobiological processes in activation and proliferation of precursor melanocytes after UV-exposure: ultrastructural differentiation of precursor melanocytes from Langerhans cells

    SciTech Connect

    Jimbow, K.; Uesugi, T.

    1982-02-01

    Photobiological processes involving new melanogenesis after exposure to ultraviolet (UV) light were experimentally studied in C57 black adult mice by histochemistry, cytochemistry, and autoradiography. The trunk and the plantar region of the foot, where no functioning melanocytes were present before exposure, were exposed to UV-A for 14 consecutive days. Both regions revealed a basically similar pattern for new melanogenesis which involved an activation of precursor melanocytes. Essentially all of ''indeterminate'' cells appeared to be precursor melanocytes, the fine structure of which could be differentiated even from poorly developed Langerhans cells. New melanogenesis was manifested by 4 stages of cellular and subcellular reactions of these cells as indicated by histochemistry of dihydroxyphenylalanine (dopa) and autoradiography of thymidine incorporation: (a) an initial lag in the activation of precursor melanocytes with development of Golgi cisternae and rough endoplasmic reticulum followed by formation of unmelanized melanosomes (day 0 to 2); (b) synthesis of active tyrosinase accumulated in Golgi cisternae and vesicles with subsequent formation of melanized melanosomes in these cells (day 3 to 5); (c) mitotic proliferation of many of these activated cells, followed by an exponential increase of new melanocytes (day 6 to 7); and (d) melanosome transfer with differentiation of 10 nm filaments and arborization of dendrites, but without any significant change in the melanocyte population (day 8 to 14). The melanosome transfer was, however, not obvious until after 7 days of exposure. The size of newly synthesized melanosomes was similar to that of tail skin where native melanocytes were present before exposure.

  18. A requirement for physical linkage between determinants recognized by helper molecules and cytotoxic T cell precursors in the induction of cytotoxic T cell responses.

    PubMed

    Krowka, J F; Singh, B; Fotedar, A; Mosmann, T; Giedlin, M A; Pilarski, L M

    1986-05-15

    It has long been understood that both antibody and delayed-type hypersensitivity responses are induced through collaborative events in which the determinants recognized by the precursor cells must be physically linked to the determinants recognized by the helper. Although it is clear that the generation of memory cytotoxic T lymphocyte precursors (CTLp) involves linked recognition of determinants, the induction of CTL responses has been viewed as being dependent upon interleukin 2 (IL 2), which could be provided by a helper cell, but independent of requirements for antigen bridging. In this work, we have designed a system that lacks exogenous IL 2 by using as our source of help, antigen-specific helper molecules derived from helper T cells. These soluble helper molecules are uncontaminated by IL 2 and unlike a helper cell, are unable to produce IL 2. Helper molecules specific for chicken red blood cells (Crbc) and for a synthetic polypeptide, poly 18, were tested. Thymocyte responders require a source of help to respond to alloantigens intrinsically expressed on the surface of adherent stimulator cells. To analyze the mechanism whereby the helper molecules acted, we used a system involving recognition of haptenic and carrier determinants that were physically linked by virtue of being located on the same cell surface (intra-structural linkage). Adherent stimulator cells were pulsed with Crbc or poly 18 so that the alloantigens recognized by the thymocyte CTLp (intrinsically expressed class I) were either linked or unlinked to the carrier determinants (Crbc or poly 18) presented by the adherent cells and recognized by the helper molecules. Both types of helper molecule were shown to be antigen-specific in crisscross experiments. The helper molecules specific for Crbc were able to induce the thymocyte CTLp only when both hapten and carrier were present on the same stimulator cell surface. Because we were not able to detect a requirement for H-2-restricted recognition

  19. Efficient perovskite solar cells fabricated using an aqueous lead nitrate precursor.

    PubMed

    Hsieh, Tsung-Yu; Wei, Tzu-Chien; Wu, Kuan-Lin; Ikegami, Masashi; Miyasaka, Tsutomu

    2015-09-04

    A novel, aqueous precursor system (Pb(NO3)2 + water) is developed to replace conventional (PbI2 + DMF) for fabricating methylammonium lead iodide (MAPbI3) perovskite solar cells (PSCs). When the morphology and surface coverage of the Pb(NO3)2 film was controlled during coating, a power conversion efficiency of 12.58% under standard conditions (AM1.5, 100 mW cm(-2)) was achieved for the PSC.

  20. The NMR study of derivatives of substituted inosine - The precursors of AICAr

    NASA Astrophysics Data System (ADS)

    Włostowski, Jacek; Krawczyk, Hanna

    2010-12-01

    In this article we describe how the simultaneous presence and interaction of the aromatic ring at position 1 of an inosine derivative with the acetyl group at positions 2, 3, 5 of the furanose ring convert conformation of the nucleoside into the major conformer in solution. During the synthesis of AICAr, we obtained products ( A and B) of inosine protected with 2,4-dinitrochlorobenzene, with two sets of NMR signals, each with very similar chemical shifts. Analysis of experimental NMR spectra and theoretical GIAO - DFT calculations were performed to obtain information about the stereochemistry of the products.

  1. The correlation between the percent of CD3- CD56+ cells and NK precursor function.

    PubMed

    Gharehbaghian, Ahmad; Donaldson, Craig; Newman, John; Bannister, Gordon; Bradley, Benjamin A

    2006-12-01

    The number and function of human natural killer (NK) cells are generally assessed to monitor the baseline of immune function, the effect of treatment, the progress of malignancy or metastases and diseases. NK cells recognise and kill target cells in the absence of prior sensitisation and are able to defend the host from infection or prevent the progression of a disease. Human NK cells express CD16 and CD56 which are (massively) being used as a major hallmark for the NK cell. The purpose of this study was to identify the unique subsets of peripheral blood mononuclear cells (PBMC) (%CD3-CD56+ cells) by flow cytometry and to determine whether there is any correlation with functionally mature progeny of (NKp) precursor after five days of culture. The correlation was analysed using samples obtained from 120 Caucasian patients. 20-30ml of whole blood was collected in sterile tube containing preservative free sodium heparin and a similar sample was obtained after five days. Maturation of NKp required the continuous presence of recombinant interleukin 2 (rIL-2), or interleukin 15 (rIL-15) and functional maturity of NK cells was determined by their ability to lyse target cells from the K562 cell line. The NK precursor frequency was measured by limiting dilution analysis (LDA), which The NKpf assay was set up with a range of cell dilutions from 40,000 to 625 per 100ml/well in 96 well culture plates. At the end of the culture period the K562 cell line labelled with Europium (Eu-K562) was added and Eu release measured in culture supernatants using time-resolved fluorometry. The PBMC were set up in parallel cultures under various conditions . On day five cells were collected from culture plates and adjusted to 1x10 cells/ml and then mixed. The mixture was incubated and anti CD3 and anti CD56 were added. NK cells were enumerated in 120 patients by double staining with a combination of anti-CD3- and anti-CD56+. The results of these Immunophenotyping studies by flow cytometry

  2. Kaempferol increases levels of coenzyme Q in kidney cells and serves as a biosynthetic ring precursor.

    PubMed

    Fernández-Del-Río, Lucía; Nag, Anish; Gutiérrez Casado, Elena; Ariza, Julia; Awad, Agape M; Joseph, Akil I; Kwon, Ohyun; Verdin, Eric; de Cabo, Rafael; Schneider, Claus; Torres, Jorge Z; Burón, María I; Clarke, Catherine F; Villalba, José M

    2017-09-01

    Coenzyme Q (Q) is a lipid-soluble antioxidant essential in cellular physiology. Patients with Q deficiencies, with few exceptions, seldom respond to treatment. Current therapies rely on dietary supplementation with Q10, but due to its highly lipophilic nature, Q10 is difficult to absorb by tissues and cells. Plant polyphenols, present in the human diet, are redox active and modulate numerous cellular pathways. In the present study, we tested whether treatment with polyphenols affected the content or biosynthesis of Q. Mouse kidney proximal tubule epithelial (Tkpts) cells and human embryonic kidney cells 293 (HEK 293) were treated with several types of polyphenols, and kaempferol produced the largest increase in Q levels. Experiments with stable isotope (13)C-labeled kaempferol demonstrated a previously unrecognized role of kaempferol as an aromatic ring precursor in Q biosynthesis. Investigations of the structure-function relationship of related flavonols showed the importance of two hydroxyl groups, located at C3 of the C ring and C4' of the B ring, both present in kaempferol, as important determinants of kaempferol as a Q biosynthetic precursor. Concurrently, through a mechanism not related to the enhancement of Q biosynthesis, kaempferol also augmented mitochondrial localization of Sirt3. The role of kaempferol as a precursor that increases Q levels, combined with its ability to upregulate Sirt3, identify kaempferol as a potential candidate in the design of interventions aimed on increasing endogenous Q biosynthesis, particularly in kidney. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Mitotic position and morphology of committed precursor cells in the zebrafish retina adapt to architectural changes upon tissue maturation.

    PubMed

    Weber, Isabell P; Ramos, Ana P; Strzyz, Paulina J; Leung, Louis C; Young, Stephen; Norden, Caren

    2014-04-24

    The development of complex neuronal tissues like the vertebrate retina requires the tight orchestration of cell proliferation and differentiation. Although the complexity of transcription factors and signaling pathways involved in retinogenesis has been studied extensively, the influence of tissue maturation itself has not yet been systematically explored. Here, we present a quantitative analysis of mitotic events during zebrafish retinogenesis that reveals three types of committed neuronal precursors in addition to the previously known apical progenitors. The identified precursor types present at distinct developmental stages and exhibit different mitotic location (apical versus nonapical), cleavage plane orientation, and morphology. Interestingly, the emergence of nonapically dividing committed bipolar cell precursors can be linked to an increase in apical crowding caused by the developing photoreceptor cell layer. Furthermore, genetic interference with neuronal subset specification induces ectopic divisions of committed precursors, underlining the finding that progressing morphogenesis can effect precursor division position.

  4. Repressor transcription factor 7-like 1 promotes adipogenic competency in precursor cells.

    PubMed

    Cristancho, Ana G; Schupp, Michael; Lefterova, Martina I; Cao, Shengya; Cohen, Daniel M; Chen, Christopher S; Steger, David J; Lazar, Mitchell A

    2011-09-27

    The identification of factors that define adipocyte precursor potential has important implications for obesity. Preadipocytes are fibroblastoid cells committed to becoming round lipid-laden adipocytes. In vitro, this differentiation process is facilitated by confluency, followed by adipogenic stimuli. During adipogenesis, a large number of cytostructural genes are repressed before adipocyte gene induction. Here we report that the transcriptional repressor transcription factor 7-like 1 (TCF7L1) binds and directly regulates the expression of cell structure genes. Depletion of TCF7L1 inhibits differentiation, because TCF7L1 indirectly induces the adipogenic transcription factor peroxisome proliferator-activated receptor γ in a manner that can be replaced by inhibition of myosin II activity. TCF7L1 is induced by cell contact in adipogenic cell lines, and ectopic expression of TCF7L1 alleviates the confluency requirement for adipocytic differentiation of precursor cells. In contrast, TCF7L1 is not induced during confluency of non-adipogenic fibroblasts, and, remarkably, forced expression of TCF7L1 is sufficient to commit non-adipogenic fibroblasts to an adipogenic fate. These results establish TCF7L1 as a transcriptional hub coordinating cell-cell contact with the transcriptional repression required for adipogenic competency.

  5. Zinc Deficiency Induces Apoptosis via Mitochondrial p53- and Caspase-Dependent Pathways in Human Neuronal Precursor Cells

    ERIC Educational Resources Information Center

    Seth, Rohit; Corniola, Rikki S.; Gower-Winter, Shannon D.; Morgan, Thomas J., Jr.; Bishop, Brian; Levenson, Cathy W.

    2015-01-01

    Previous studies have shown that zinc deficiency leads to apoptosis of neuronal precursor cells in vivo and in vitro. In addition to the role of p53 as a nuclear transcription factor in zinc deficient cultured human neuronal precursors (NT-2), we have now identified the translocation of phosphorylated p53 to the mitochondria and p53-dependent…

  6. Neuroprotective effect of buyang huanwu decoction on rat ischemic/reperfusion brain damage by promoting migration of neural precursor cells.

    PubMed

    Kong, Xiangying; Su, Xiaohui; Zhu, Jia; Wang, Jianzhu; Wan, Hongye; Zhong, Micun; Li, Li; Lin, Na

    2014-06-01

    Buyang Huanwu Decoction (BYHWD) is a classic formula widely used for treating stroke-induced disability, the highest morbidity of neurological disorders in China. However, the mechanism of its neuroprotection has not been fully clarified. Previous reports indicated that BYHWD may promote growth and differentiation of neural precursor cells (NPCs). The present study focused on the effects of BYHWD on migration of NPCs in rats with middle cerebral artery occlusion (MCAO). Rats were treated with different doses of BYHWD (12 and 24 grams/kg) from day 1 to day 21 after model building. BYHWD could increase the survival rate and decrease neurological scores and infarct volume as compared with the vehicle-treated MCAO rats. Moreover, BYHWD treatment significantly increased 5-bromo-2-deoxyuridine (BrdU)-positive cells in the subventricular zone (SVZ), subgranular zone (SGZ), and corpus striatum (CS) of the infarct brain. Interestingly, BYHWD could markedly enhance BrdU(+)/doublecortin(+) cells not only in the SVZ and SGZ but also in CS, by up-regulating the protein expression of migration activators, including stromal cell derived factor-1, CXC chemokine receptor 4, vascular endothelial growth factor, Reelin, and brain-derived neurotrophic factor in the ipsilateral infarct area after MCAO. In addition, BYHWD treatment was able to promote the neuronal differentiation, which was closely related to the migratory process of NPCs in MCAO rats. These findings offer evidence for the first time that BYHWD may exert its neuroprotective effects partially by promotion of NPCs migration to ischemic brain areas.

  7. Epidermal growth factor precursor in mouse lactating mammary gland alveolar cells

    SciTech Connect

    Brown, C.F.; Teng, C.T.; Pentecost, B.T.; DiAugustine, R.P. )

    1989-07-01

    Previous studies have demonstrated that high levels of epidermal growth factor (EGF) occur in human and rodent milk and that oral administration of this polypeptide stimulates rodent gastrointestinal development. It is not known whether EGF in milk originates from cells of the lactating mammary gland or is sequestered from an extramammary source. In the present study, prepro-EGF mRNA (approximately 4.7 kilobases) was detected in the CD-1 mouse mammary gland throughout the period of lactation; by comparison, negligible levels of this EGF transcript were found in the gland during pregnancy. Low levels of EGF immunoreactivity (4-5 ng/g wet wt tissue) were extracted from lactating (day 18) mammary glands with dilute acetic acid. Immunolocalization was evident with antisera to either EGF or two other regions of the EGF precursor in essentially all alveolar cells of the lactating gland. The most prominent staining with antiserum to EGF was observed along the luminal borders of cells; this pattern of cellular staining required proteolytic pretreatment of tissue sections. Western blot analyses of cell membranes isolated from the day 16 lactating mammary gland revealed an EGF-immunoreactive band at about 145K, which was equivalent in size to the EGF precursor found in mouse kidney cell membranes. Despite these findings, labeling of lactating mammary gland mince with L-(35S)methionine and cysteine for up to 4 h did not reveal any specific bands in immunoprecipitates. These cumulative findings suggest that the precursor form of EGF occurs in alveolar cells of lactating mammary gland and that this protein is translocated to the cell membrane.

  8. The Crosstalk between Myeloid Derived Suppressor Cells and Immune Cells: To Establish Immune Tolerance in Transplantation

    PubMed Central

    Wang, Shuo; Yang, Cheng

    2016-01-01

    Myeloid derived suppressor cells (MDSCs) are a heterogeneous population of myeloid precursor and progenitor cells and endowed with a robust immunosuppressive activity in multiple pathophysiological conditions. Recent studies have uncovered the crosstalk between MDSCs and immune cells (i.e., natural killer cells, dendritic cells, macrophages, natural killer T cells, and regulatory T cells) and its role in the establishment and maintenance of immune tolerant microenvironment in transplantation. Considering their strong immunosuppressive capability, MDSCs could become a prospective clinical regimen during transplantation tolerance induction, resulting in long-term graft survival with decreased or without immunosuppressive drugs. The review summarized recent research advances in this field and looked ahead at the research directions in the future. PMID:27868073

  9. Aqueous Ln(III) Luminescence Agents Derived from a Tasty Precursor

    SciTech Connect

    Jocher, C.J.; Moore, E.G.; Pierce, J.D.; Raymond, K.N.

    2008-06-02

    The synthesis, aqueous stability and photophysical properties are reported for a novel tetradentate ligand derived from maltol, a commonly used flavor enhancer. In aqueous solution, this chelate forms stable complexes with Ln(III) cations, and sensitized emission was observed from Eu(III), Yb(III), and Nd(III). A comparison with recently reported and structurally analogous ligands reveals a slightly higher basicity but lower complex stability with Eu(III) [pEu = 14.7 (1)]. A very poor metal centered quantum yield with Eu(III) was observed ({Phi}{sub tot} = 0.04%), which can be rationalized by the similar energy of the ligand triplet state and the Eu(III) {sup 5}D{sub 0} emissive level. Instead, sensitized emission from the Yb(III) and Nd(III) cations was observed, which emit in the Near Infra-Red (NIR).

  10. β-Amyloid precursor protein: function in stem cell development and Alzheimer's disease brain.

    PubMed

    Small, David H; Hu, Yanling; Bolós, Marta; Dawkins, Edgar; Foa, Lisa; Young, Kaylene M

    2014-01-01

    Stem cell therapy may be a suitable approach for the treatment of many neurodegenerative diseases. However, one major impediment to the development of successful cell-based therapies is our limited understanding of the mechanisms that instruct neural stem cell behaviour, such as proliferation and cell fate specification. The β-amyloid precursor protein (APP) of Alzheimer's disease (AD) may play an important role in neural stem cell proliferation and differentiation. Our recent work shows that in vitro, APP stimulates neural stem or progenitor cell proliferation and neuronal differentiation. The effect on proliferation is mediated by an autocrine factor that we have identified as cystatin C. As cystatin C expression is also reported to inhibit the development of amyloid pathology in APP transgenic mice, our finding has implications for the possible use of cystatin C for the therapy of AD.

  11. Double minute chromosomes can be produced from precursors derived from a chromosomal deletion.

    PubMed Central

    Carroll, S M; DeRose, M L; Gaudray, P; Moore, C M; Needham-Vandevanter, D R; Von Hoff, D D; Wahl, G M

    1988-01-01

    Recent experiments have shown that gene amplification can be mediated by submicroscopic, autonomously replicating, circular extrachromosomal molecules. We refer to those molecules as episomes (S. Carroll, P. Gaudray, M. L. DeRose, J. F. Emery, J. L. Meinkoth, E. Nakkim, M. Subler, D. D. Von Hoff, and G. M. Wahl, Mol. Cell. Biol. 7:1740-1750, 1987). The experiments reported in this paper explore the way episomes are formed and their fate in the cell over time. The data reveal that in our system the episomes are initially 250 kilobases, but gradually enlarge until they become double minute chromosomes. In addition, we show that episomes or double minute chromosomes can integrate into chromosomes. Our results also suggest that episomes can be produced by deletion of the corresponding sequences from the chromosome. Images PMID:2898098

  12. Bone marrow-derived lung epithelial cells.

    PubMed

    Krause, Diane S

    2008-08-15

    Bone marrow-derived cells can take on the phenotype of epithelial cells and express epithelial-specific genes in multiple organs. Here, we focus on recent data on the appearance of marrow-derived epithelial cells in the adult lung. These findings have garnered significant skepticism because in most cases marrow-derived epithelial cells are very rare, the marrow cell of origin is not known, the techniques for detection have needed improvement, and there seem to be multiple mechanisms by which this occurs. Recent studies have focused on these concerns. Once these important concerns are addressed, further studies on the function(s) of these cells will need to be performed to determine whether this engraftment has any clinical significance-either beneficial or detrimental.

  13. Using superoxide dismutase/catalase mimetics to manipulate the redox environment of neural precursor cells.

    PubMed

    Limoli, C L; Giedzinski, E; Baure, J; Doctrow, S R; Rola, R; Fike, J R

    2006-01-01

    Past work has shown that neural precursor cells are predisposed to redox sensitive changes, and that oxidative stress plays a critical role in the acute and persistent changes that occur within the irradiated CNS. Irradiation leads to a marked rise in reactive oxygen species (ROS) that correlates with oxidative endpoints in vivo and reductions in neurogenesis. To better understand the impact of oxidative stress on neural precursor cells, and to determine if radiation-induced oxidative damage and precursor cell loss after irradiation could be reduced, a series of antioxidant compounds (EUK-134, EUK-163, EUK-172, EUK-189) were tested, three of which possess both superoxide dismutase (SOD) and catalase activities and one (EUK-163) whose only significant activity is SOD. Our results show that these SOD/catalase mimetics apparently increase the oxidation of a ROS-sensitive fluorescent indicator dye, particularly after short (12 h) treatments, but that longer treatments (24 h) decrease oxidation attributable to radiation-induced ROS. Similarly, other studies found that cells incubated with CuZnSOD showed some increase in intracellular ROS levels. Subsequent data suggested that the dye-oxidising capabilities of the EUK compounds were linked to differences in their catalase activity and, most likely, their ability to catalyse peroxidative pathways. In unirradiated mice, the EUK-134 analogue induced some decrease of proliferating precursor cells and immature neurons 48 h after radiation, an effect that may be attributable to cytotoxicity and/or inhibition of precursor proliferation. In irradiated mice, a single injection of EUK-134 was not found to be an effective radioprotector at acute times (48 h). The present results support continued development of our in vitro model as a tool for predicting certain in vivo responses, and suggest that in some biological systems the capability to scavenge superoxide but produce excess H(2)O(2), as is known for CuZnSOD, may be

  14. Blood-brain barrier promotes differentiation of human fetal neural precursor cells.

    PubMed

    Chintawar, Satyan; Cayrol, Romain; Antel, Jack; Pandolfo, Massimo; Prat, Alexandre

    2009-04-01

    In the stem cell niche, neural stem cells (NSCs) are in close contact with the specialized blood-brain barrier (BBB) endothelial cells (ECs) that modulate their proliferation and differentiation behavior. NSCs are also an attractive source for cell transplantation and neural tissue repair after central nervous system injury. After systemic grafting, they are confronted with the BBB before they can enter the brain parenchyma. We investigated the interactions of human fetal neural precursor cells (hfNPCs) with human brain ECs in an in vitro model using primary cultures. We demonstrated that hfNPCs efficiently differentiate to neurons, astrocytes, and oligodendrocytes and move to the subendothelial space of human BBB endothelium, but not to pulmonary artery ECs. Effective differentiation was found to be dependent on the chemokine CCL2/MCP-1, but not on CXCL8/IL-8. Our findings suggest that neural precursor cells specifically interact with the BBB endothelium and differentiate in the subendothelial niche into astrocytes, neurons, and oligodendrocytes, under the influence of the chemokine CCL2/MCP-1.

  15. Repressor transcription factor 7-like 1 promotes adipogenic competency in precursor cells

    PubMed Central

    Cristancho, Ana G.; Schupp, Michael; Lefterova, Martina I.; Cao, Shengya; Cohen, Daniel M.; Chen, Christopher S.; Steger, David J.; Lazar, Mitchell A.

    2011-01-01

    The identification of factors that define adipocyte precursor potential has important implications for obesity. Preadipocytes are fibroblastoid cells committed to becoming round lipid-laden adipocytes. In vitro, this differentiation process is facilitated by confluency, followed by adipogenic stimuli. During adipogenesis, a large number of cytostructural genes are repressed before adipocyte gene induction. Here we report that the transcriptional repressor transcription factor 7-like 1 (TCF7L1) binds and directly regulates the expression of cell structure genes. Depletion of TCF7L1 inhibits differentiation, because TCF7L1 indirectly induces the adipogenic transcription factor peroxisome proliferator-activated receptor γ in a manner that can be replaced by inhibition of myosin II activity. TCF7L1 is induced by cell contact in adipogenic cell lines, and ectopic expression of TCF7L1 alleviates the confluency requirement for adipocytic differentiation of precursor cells. In contrast, TCF7L1 is not induced during confluency of non-adipogenic fibroblasts, and, remarkably, forced expression of TCF7L1 is sufficient to commit non-adipogenic fibroblasts to an adipogenic fate. These results establish TCF7L1 as a transcriptional hub coordinating cell–cell contact with the transcriptional repression required for adipogenic competency. PMID:21914845

  16. A Critical Precursor Frequency of Donor-Reactive CD4+ T Cell Help is Required for CD8+ T Cell-Mediated CD28/CD154-Independent Rejection

    PubMed Central

    Ford, Mandy L.; Wagener, Maylene E.; Hanna, Samantha S.; Pearson, Thomas C.; Kirk, Allan D.; Larsen, Christian P.

    2008-01-01

    Antigen-specific precursor frequency is increasingly being appreciated as an important factor in determining the kinetics, magnitude, and degree of differentiation of T cell responses, and recently was found to play a critical role in determining the relative requirement of CD8+ T cells for CD28- and CD154-mediated costimulatory signals during transplantation. We addressed the possibility that variations in CD4+ T cell precursor frequency following transplantation might affect CD4+ T cell proliferation, effector function, and provision of help for donor-reactive B cell and CD8+ T cell responses. Using a transgenic model system wherein increasing frequencies of donor-reactive CD4+ T cells were transferred into skin graft recipients, we observed that a critical CD4+ T cell threshold precursor frequency was necessary to provide help following blockade of the CD28 and CD154 costimulatory pathways, as measured by increased B cell and CD8+ T cell responses and precipitation of graft rejection. In contrast to high-frequency CD8+ T cell responses, this effect was observed even though the proliferative and cytokine responses of Ag-specific CD4+ T cells were inhibited. Thus, we conclude that an initial high frequency of donor-reactive CD4+ T cells uncouples T cell proliferative and effector cytokine production from the provision of T cell help. This is an author-produced version of a manuscript accepted for publication in The Journal of Immunology (The JI). The American Association of Immunologists, Inc. (AAI), publisher of The JI, holds the copyright to this manuscript. This version of the manuscript has not yet been copyedited or subjected to editorial proofreading by The JI; hence, it may differ from the final version published in The JI (online and in print). AAI (The JI) is not liable for errors or omissions in this author-produced version of the manuscript or in any version derived from it by the U.S. National Institutes of Health or any other third party. The final

  17. Sphedgehog is expressed by pigment cell precursors during early gastrulation in Strongylocentrotus purpuratus.

    PubMed

    Egaña, Ana L; Ernst, Susan G

    2004-10-01

    We have sequenced the Sphedgehog (Sphh) gene from the sea urchin Strongylocentrotus purpuratus. Sphh transcripts are detected first at the mesenchyme blastula stage, and they accumulate throughout early embryogenesis. The Sphh protein is produced by precursor pigment cells during early and midgastrulation. NiCl2 inhibits pigment cell differentiation in sea urchins. Here, we show that, in S. purpuratus, nickel affects a process(es) between 17 and 24 hr of development, corresponding to the time period when Sphh mRNA is first detected. However, nickel treatment does not alter the early expression of Sphh.

  18. Characterization of nonlymphoid cells derived from rat peripheral lymph

    PubMed Central

    Pugh, CW; MacPherson, GG; Steer, HW

    1983-01-01

    Mesenteric lymphadenectomy in rats is followed by union of peripheral and central lymphatics, allowing the collection of intestine-derived peripheral lymph cells via the thoracic duct for several days. These cells include a proportion of nonlymphoid cells (NLC) that show irregular and heterogeneous surface morphology including long pseudopodia and veils. They stain variably for nonspecific esterase and acid phosphatase and are ATPase-positive. Their nuclei are irregular and some contain cytoplasmic inclusions, some of which show peroxidase activity and/or contain DNA. NLC have a range of densitites generally lower than that of lymphocytes. Freshly collected NLC express the leukocyte-common antigen (defined by monoclonal antibody MRC Ox 1) and Ia antigens (I-A and I-E subregion products defined by monoclonal antibodies) but they show a relative lack of other surface markers normally found on rat B or T lymphocytes (W3/13, W3/25, MRC Ox 12 (sIg), MRC Ox 19) or rat macrophages (FcR, C’R, mannose R, W3/25). In general NLC are only weakly adherent to glass or plastic. Although a subpopulation of NLC appear to have had a phagocytic past, freshly collected NLC fail to phagocytose a variety of test particles in vitro. NLC also appear incapable of pinocytosis in vitro. This heterogeneity may represent distinct subpopulations of NLC or different stages in the development of a single cell lineage. Direct cannulation of mesenteric lacteals shows that the majority of NLC are derived from the small intestine and their precursors appear to be present both in lamina propria and Peyer's patches. Kinetic studies, following irradiation or intravenous tritiated thymidine, show that the majority of NLC turn over rapidly in the intestine with a modal time of 3-5 d. Studies with bone marrow chimeras show that they are derived from a rapidly dividing precursor present in normal bone marrow. NLC occur at very low frequencies in normal thoracic duct lymph at all times following

  19. Isolation and characterization of bone marrow-derived mesenchymal progenitor cells with myogenic and neuronal properties.

    PubMed

    Shiota, Mitsutaka; Heike, Toshio; Haruyama, Munetada; Baba, Shiro; Tsuchiya, Atsunori; Fujino, Hisanori; Kobayashi, Hirohiko; Kato, Takeo; Umeda, Katsutsugu; Yoshimoto, Momoko; Nakahata, Tatsutoshi

    2007-03-10

    Sphere formation has been utilized as a way to isolate multipotent stem/progenitor cells from various tissues. However, very few studies on bone marrow-derived spheres have been published and assessed their multipotentiality. In this study, multipotent marrow cell populations were isolated using a three-step method. First, after elimination of hematopoietic cells, murine marrow-derived adherent cells were cultured in plastic dishes until small cells gradually appeared and multiplied. Cells were then cultured under non-adherent conditions and formed spheres that were immunopositive for a neural precursor marker, nestin. RT-PCR analysis also revealed that the spheres were positive for nestin in addition to PPARgamma, osf2, SOX9, and myoD, which are markers of precursors of adipocytic, osteoblastic, chondrocytic, and skeletal myeloblastic lineages, respectively. Finally, spheres were dissociated into single cells and expanded in adherent cultures. Under appropriate induction conditions, the sphere-derived cells acquired the phenotypic properties in vitro of neurons, skeletal myoblasts, and beating cardiomyocytes, as well as adipocytes, osteoblasts, and chondrocytes. Next, sphere-derived cells were transplanted into murine myocardial infarction models. One month later, they had become engrafted as cardiomyocytes, and cardiac catheterization showed significant functional improvements. Thus, sphere-derived cells represent a new approach to enhance the multi-differentiation potential of murine bone marrow.

  20. Comparison of naïve and central memory derived CD8+ effector cell engraftment fitness and function following adoptive transfer

    PubMed Central

    Wang, Xiuli; Wong, ChingLam W.; Urak, Ryan; Taus, Ellie; Aguilar, Brenda; Chang, Wen-Chung; Mardiros, Armen; Budde, Lihua E.; Brown, Christine E.; Berger, Carolina; Forman, Stephen J.; Jensen, Michael C.

    2016-01-01

    abstract Human CD8+ effector T cells derived from CD45RO+CD62L+ precursors enriched for central memory (TCM) precursors retain the capacity to engraft and reconstitute functional memory upon adoptive transfer, whereas effectors derived from CD45RO+CD62L− precursors enriched for effector memory precursors do not. Here we sought to compare the engraftment fitness and function of CD8+ effector T cells derived from CD45RA+CD62L+ precursors enriched for naïve and stem cell memory precursors (TN/SCM) with that of TCM. We found that cytotoxic T cells (CTLs) derived from TCM transcribed higher levels of CD28, FOS, INFγ, Eomesodermin (Eomes), and lower levels of BCL2L11, maintained higher levels of phosphorylated AKT, and displayed enhanced sensitivity to the proliferative and anti-apoptotic effects of γ-chain cytokines compared to CTLs derived from TN/SCM. Higher frequencies of CTLs derived from TCM retained CD28 expression and upon activation secreted higher levels of IL-2. In NOD/Scid IL-2RγCnull mice, CD8+ TCM derived CTLs engrafted to higher frequencies in response to human IL-15 and mounted robust proliferative responses to an immunostimulatory vaccine. Similarly, CD8+ TCM derived CD19CAR+ CTLs exhibited superior antitumor potency following adoptive transfer compared to their CD8+ TN/SCM derived counterparts. These studies support the use of TCM enriched cell products for adoptive therapy of cancer. PMID:26942092

  1. Comparison of naïve and central memory derived CD8(+) effector cell engraftment fitness and function following adoptive transfer.

    PubMed

    Wang, Xiuli; Wong, ChingLam W; Urak, Ryan; Taus, Ellie; Aguilar, Brenda; Chang, Wen-Chung; Mardiros, Armen; Budde, Lihua E; Brown, Christine E; Berger, Carolina; Forman, Stephen J; Jensen, Michael C

    Human CD8(+) effector T cells derived from CD45RO(+)CD62L(+) precursors enriched for central memory (TCM) precursors retain the capacity to engraft and reconstitute functional memory upon adoptive transfer, whereas effectors derived from CD45RO(+)CD62L(-) precursors enriched for effector memory precursors do not. Here we sought to compare the engraftment fitness and function of CD8(+) effector T cells derived from CD45RA(+)CD62L(+) precursors enriched for naïve and stem cell memory precursors (TN/SCM) with that of TCM. We found that cytotoxic T cells (CTLs) derived from TCM transcribed higher levels of CD28, FOS, INFγ, Eomesodermin (Eomes), and lower levels of BCL2L11, maintained higher levels of phosphorylated AKT, and displayed enhanced sensitivity to the proliferative and anti-apoptotic effects of γ-chain cytokines compared to CTLs derived from TN/SCM. Higher frequencies of CTLs derived from TCM retained CD28 expression and upon activation secreted higher levels of IL-2. In NOD/Scid IL-2RγC(null) mice, CD8(+) TCM derived CTLs engrafted to higher frequencies in response to human IL-15 and mounted robust proliferative responses to an immunostimulatory vaccine. Similarly, CD8(+) TCM derived CD19CAR(+) CTLs exhibited superior antitumor potency following adoptive transfer compared to their CD8(+) TN/SCM derived counterparts. These studies support the use of TCM enriched cell products for adoptive therapy of cancer.

  2. Synergic Effect between Adsorption and Photocatalysis of Metal-Free g-C3N4 Derived from Different Precursors

    PubMed Central

    Xu, Huan-Yan; Wu, Li-Cheng; Zhao, Hang; Jin, Li-Guo; Qi, Shu-Yan

    2015-01-01

    Graphitic carbon nitride (g-C3N4) used in this work was obtained by heating dicyandiamide and melamine, respectively, at different temperatures. The differences of g-C3N4 derived from different precursors in phase composition, functional group, surface morphology, microstructure, surface property, band gap and specific surface area were investigated by X-ray diffraction, Fourier transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, X-ray photoelectron spectroscopy, UV-visible diffuse reflection spectroscopy and BET surface area analyzer, respectively. The photocatalytic discoloration of an active cationic dye, Methylene Blue (MB) under visible-light irradiation indicated that g-C3N4 derived from melamine at 500°C (CN-M500) had higher adsorption capacity and better photocatalytic activity than that from dicyandiamide at 500°C (CN-D500), which was attributed to the larger surface area of CN-M500. MB discoloration ratio over CN-M500 was affected by initial MB concentration and photocatalyst dosage. After 120 min reaction time, the blue color of MB solution disappeared completely. Subsequently, based on the measurement of the surface Zeta potentials of CN-M500 at different pHs, an active anionic dye, Methyl Orange (MO) was selected as the contrastive target pollutant with MB to reveal the synergic effect between adsorption and photocatalysis. Finally, the photocatalytic mechanism was discussed. PMID:26565712

  3. Synergic Effect between Adsorption and Photocatalysis of Metal-Free g-C3N4 Derived from Different Precursors.

    PubMed

    Xu, Huan-Yan; Wu, Li-Cheng; Zhao, Hang; Jin, Li-Guo; Qi, Shu-Yan

    2015-01-01

    Graphitic carbon nitride (g-C3N4) used in this work was obtained by heating dicyandiamide and melamine, respectively, at different temperatures. The differences of g-C3N4 derived from different precursors in phase composition, functional group, surface morphology, microstructure, surface property, band gap and specific surface area were investigated by X-ray diffraction, Fourier transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, X-ray photoelectron spectroscopy, UV-visible diffuse reflection spectroscopy and BET surface area analyzer, respectively. The photocatalytic discoloration of an active cationic dye, Methylene Blue (MB) under visible-light irradiation indicated that g-C3N4 derived from melamine at 500°C (CN-M500) had higher adsorption capacity and better photocatalytic activity than that from dicyandiamide at 500°C (CN-D500), which was attributed to the larger surface area of CN-M500. MB discoloration ratio over CN-M500 was affected by initial MB concentration and photocatalyst dosage. After 120 min reaction time, the blue color of MB solution disappeared completely. Subsequently, based on the measurement of the surface Zeta potentials of CN-M500 at different pHs, an active anionic dye, Methyl Orange (MO) was selected as the contrastive target pollutant with MB to reveal the synergic effect between adsorption and photocatalysis. Finally, the photocatalytic mechanism was discussed.

  4. Lack of effect of a granulocyte proliferation inhibitor or their committed precursor cells.

    PubMed

    Lord, B I; Testa, N G; Wright, E G; Banerjee, R K

    1977-05-01

    Using the agar culture technique, we have measured the effect of granulocyte extracts GCE (and of erythrocyte-RCE and lymph node extracts-LNE) on the growth and proliferation of the committed granulocytic precursor cells, CFU-C. In addition we have determined their effects on the proliferation of the developing colony cells and on the ultimate cell production in the colonies. The results show that GCE has no effect on the growth or proliferative activity on the CFU-C. It does, however, reduce both the autoradiographic labelling indices of the developing colony cells and the net colony cellularities, acting as a cell cycle modulator. These are effects specific to the GCE since at the dose levels used, neither RCE nor LNE affected these measurements.

  5. Group 13 β-ketoiminate compounds: gallium hydride derivatives as molecular precursors to thin films of Ga2O3.

    PubMed

    Pugh, David; Marchand, Peter; Parkin, Ivan P; Carmalt, Claire J

    2012-06-04

    Bis(β-ketoimine) ligands, [R{N(H)C(Me)-CHC(Me)═O}(2)] (L(1)H(2), R = (CH(2))(2); L(2)H(2), R = (CH(2))(3)), linked by ethylene (L(1)) and propylene (L(2)) bridges have been used to form aluminum, gallium, and indium chloride complexes [Al(L(1))Cl] (3), [Ga(L(n))Cl] (4, n = 1; 6, n = 2) and [In(L(n))Cl] (5, n = 1; 7, n = 2). Ligand L(1) has also been used to form a gallium hydride derivative [Ga(L(1))H] (8), but indium analogues could not be made. β-ketoimine ligands, [Me(2)N(CH(2))(3)N(H)C(R')-CHC(R')═O] (L(3)H, R' = Me; L(4)H, R' = Ph), with a donor-functionalized Lewis base have also been synthesized and used to form gallium and indium alkyl complexes, [Ga(L(3))Me(2)] (9) and [In(L(3))Me(2)] (10), which were isolated as oils. The related gallium hydride complexes, [Ga(L(n))H(2)] (11, n = 3; 12, n = 4), were also prepared, but again no indium hydride species could be made. The complexes were characterized mainly by NMR spectroscopy, mass spectrometry, and single crystal X-ray diffraction. The β-ketoiminate gallium hydride compounds (8 and 11) have been used as single-source precursors for the deposition of Ga(2)O(3) by aerosol-assisted (AA)CVD with toluene as the solvent. The quality of the films varied according to the precursor used, with the complex [Ga(L(1))H] (8) giving by far the best quality films. Although the films were amorphous as deposited, they could be annealed at 1000 °C to form crystalline Ga(2)O(3). The films were analyzed by powder XRD, SEM, and EDX.

  6. Susceptibility of Human Embryonic Stem Cell-Derived Neural Cells to Japanese Encephalitis Virus Infection

    PubMed Central

    Shen, Shih-Cheng; Shen, Ching-I; Lin, Ho; Chen, Chun-Jung; Chang, Chia-Yu; Chen, Sheng-Mei; Lee, Hsiu-Chin; Lai, Ping-Shan; Su, Hong-Lin

    2014-01-01

    Pluripotent human embryonic stem cells (hESCs) can be efficiently directed to become immature neuroepithelial precursor cells (NPCs) and functional mature neural cells, including neurotransmitter-secreting neurons and glial cells. Investigating the susceptibility of these hESCs-derived neural cells to neurotrophic viruses, such as Japanese encephalitis virus (JEV), provides insight into the viral cell tropism in the infected human brain. We demonstrate that hESC-derived NPCs are highly vulnerable to JEV infection at a low multiplicity of infection (MOI). In addition, glial fibrillary acid protein (GFAP)-expressing glial cells are also susceptible to JEV infection. In contrast, only a few mature neurons were infected at MOI 10 or higher on the third day post-infection. In addition, functional neurotransmitter-secreting neurons are also resistant to JEV infection at high MOI. Moreover, we discover that vimentin intermediate filament, reported as a putative neurovirulent JEV receptor, is highly expressed in NPCs and glial cells, but not mature neurons. These results indicate that the expression of vimentin in neural cells correlates to the cell tropism of JEV. Finally, we further demonstrate that membranous vimentin is necessary for the susceptibility of hESC-derived NPCs to JEV infection. PMID:25517725

  7. Diethanolamine Alters Proliferation and Choline Metabolism in Mouse Neural Precursor Cells

    PubMed Central

    Niculescu, Mihai D.; Wu, Renan; Guo, Zhong; da Costa, Kerry Ann; Zeisel, Steven H.

    2008-01-01

    Diethanolamine (DEA) is a widely used ingredient in many consumer products and in a number of industrial applications. It has been previously reported that dermal administration of DEA to mice diminished hepatic stores of choline and altered brain development in the fetus. The aim of this study was to use mouse neural precursor cells in vitro to assess the mechanism underlying the effects of DEA. Cells exposed to DEA treatment (3mM) proliferated less (by 5-bromo-2-deoxyuridine incorporation) at 48 h (24% of control [CT]), and had increased apoptosis at 72 h (308% of CT). Uptake of choline into cells was reduced by DEA treatment (to 52% of CT), resulting in diminished intracellular concentrations of choline and phosphocholine (55 and 12% of CT, respectively). When choline concentration in the growth medium was increased threefold (to 210μM), the effects of DEA exposure on cell proliferation and apoptosis were prevented, however, intracellular phosphocholine concentrations remained low. In choline kinase assays, we observed that DEA can be phosphorylated to phospho-DEA at the expense of choline. Thus, the effects of DEA are likely mediated by inhibition of choline transport into neural precursor cells and by altered metabolism of choline. Our study suggests that prenatal exposure to DEA may have a detrimental effect on brain development. PMID:17204582

  8. Anti-CD22–chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia

    PubMed Central

    Haso, Waleed; Lee, Daniel W.; Shah, Nirali N.; Stetler-Stevenson, Maryalice; Yuan, Constance M.; Pastan, Ira H.; Dimitrov, Dimiter S.; Morgan, Richard A.; FitzGerald, David J.; Barrett, David M.; Wayne, Alan S.; Mackall, Crystal L.

    2013-01-01

    Immune targeting of B-cell malignancies using chimeric antigen receptors (CARs) is a promising new approach, but critical factors impacting CAR efficacy remain unclear. To test the suitability of targeting CD22 on precursor B-cell acute lymphoblastic leukemia (BCP-ALL), lymphoblasts from 111 patients with BCP-ALL were assayed for CD22 expression and all were found to be CD22-positive, with median CD22 expression levels of 3500 sites/cell. Three distinct binding domains targeting CD22 were fused to various TCR signaling domains ± an IgG heavy chain constant domain (CH2CH3) to create a series of vector constructs suitable to delineate optimal CAR configuration. CARs derived from the m971 anti-CD22 mAb, which targets a proximal CD22 epitope demonstrated superior antileukemic activity compared with those incorporating other binding domains, and addition of a 4-1BB signaling domain to CD28.CD3ζ constructs diminished potency, whereas increasing affinity of the anti-CD22 binding motif, and extending the CD22 binding domain away from the membrane via CH2CH3 had no effect. We conclude that second-generation m971 mAb-derived anti-CD22 CARs are promising novel therapeutics that should be tested in BCP-ALL. PMID:23243285

  9. Effects of Rho Kinase Inhibitors on Grafts of Dopaminergic Cell Precursors in a Rat Model of Parkinson's Disease.

    PubMed

    Rodriguez-Pallares, Jannette; Rodriguez-Perez, Ana I; Muñoz, Ana; Parga, Juan A; Toledo-Aral, Juan J; Labandeira-Garcia, Jose L

    2016-06-01

    excellent strategy for improving survival of grafted neurons and graft-derived dopaminergic innervation. The present results discourage the treatment of suspensions of dopaminergic precursors with ROCK inhibitors in the pregrafting period. However, treatment of the host (patients with PD) with ROCK inhibitors, currently used against vascular diseases, may be a useful adjuvant to cell therapy in PD. ©AlphaMed Press.

  10. Effects of Rho Kinase Inhibitors on Grafts of Dopaminergic Cell Precursors in a Rat Model of Parkinson’s Disease

    PubMed Central

    Rodriguez-Pallares, Jannette; Rodriguez-Perez, Ana I.; Muñoz, Ana; Parga, Juan A.; Toledo-Aral, Juan J.

    2016-01-01

    research and may be an excellent strategy for improving survival of grafted neurons and graft-derived dopaminergic innervation. The present results discourage the treatment of suspensions of dopaminergic precursors with ROCK inhibitors in the pregrafting period. However, treatment of the host (patients with PD) with ROCK inhibitors, currently used against vascular diseases, may be a useful adjuvant to cell therapy in PD. PMID:27075764

  11. The planarian nanos-like gene Smednos is expressed in germline and eye precursor cells during development and regeneration.

    PubMed

    Handberg-Thorsager, Mette; Saló, Emili

    2007-05-01

    Planarians are highly regenerative organisms with the ability to remake all their cell types, including the germ cells. The germ cells have been suggested to arise from totipotent neoblasts through epigenetic mechanisms. Nanos is a zinc-finger protein with a widely conserved role in the maintenance of germ cell identity. In this work, we describe the expression of a planarian nanos-like gene Smednos in two kinds of precursor cells namely, primordial germ cells and eye precursor cells, during both development and regeneration of the planarian Schmidtea mediterranea. In sexual planarians, Smednos is expressed in presumptive male primordial germ cells of embryos from stage 8 of embryogenesis and throughout development of the male gonads and in the female primordial germ cells of the ovary. Thus, upon hatching, juvenile planarians do possess primordial germ cells. In the asexual strain, Smednos is expressed in presumptive male and female primordial germ cells. During regeneration, Smednos expression is maintained in the primordial germ cells, and new clusters of Smednos-positive cells appear in the regenerated tissue. Remarkably, during the final stages of development (stage 8 of embryogenesis) and during regeneration of the planarian eye, Smednos is expressed in cells surrounding the differentiating eye cells, possibly corresponding to eye precursor cells. Our results suggest that similar genetic mechanisms might be used to control the differentiation of precursor cells during development and regeneration in planarians.

  12. Intestinal label-retaining cells are secretory precursors expressing Lgr5.

    PubMed

    Buczacki, Simon J A; Zecchini, Heather Ireland; Nicholson, Anna M; Russell, Roslin; Vermeulen, Louis; Kemp, Richard; Winton, Douglas J

    2013-03-07

    The rapid cell turnover of the intestinal epithelium is achieved from small numbers of stem cells located in the base of glandular crypts. These stem cells have been variously described as rapidly cycling or quiescent. A functional arrangement of stem cells that reconciles both of these behaviours has so far been difficult to obtain. Alternative explanations for quiescent cells have been that they act as a parallel or reserve population that replace rapidly cycling stem cells periodically or after injury; their exact nature remains unknown. Here we show mouse intestinal quiescent cells to be precursors that are committed to mature into differentiated secretory cells of the Paneth and enteroendocrine lineage. However, crucially we find that after intestinal injury they are capable of extensive proliferation and can give rise to clones comprising the main epithelial cell types. Thus, quiescent cells can be recalled to the stem-cell state. These findings establish quiescent cells as an effective clonogenic reserve and provide a motivation for investigating their role in pathologies such as colorectal cancers and intestinal inflammation.

  13. Metabolic Characterization of Intact Cells Reveals Intracellular Amyloid Beta but Not Its Precursor Protein to Reduce Mitochondrial Respiration.

    PubMed

    Schaefer, Patrick M; von Einem, Bjoern; Walther, Paul; Calzia, Enrico; von Arnim, Christine A F

    2016-01-01

    One hallmark of Alzheimer´s disease are senile plaques consisting of amyloid beta (Aβ), which derives from the processing of the amyloid precursor protein (APP). Mitochondrial dysfunction has been linked to the pathogenesis of Alzheimer´s disease and both Aβ and APP have been reported to affect mitochondrial function in isolated systems. However, in intact cells, considering a physiological localization of APP and Aβ, it is pending what triggers the mitochondrial defect. Thus, the aim of this study was to dissect the impact of APP versus Aβ in inducing mitochondrial alterations with respect to their subcellular localization. We performed an overexpression of APP or beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), increasing APP and Aβ levels or Aβ alone, respectively. Conducting a comprehensive metabolic characterization we demonstrate that only APP overexpression reduced mitochondrial respiration, despite lower extracellular Aβ levels compared to BACE overexpression. Surprisingly, this could be rescued by a gamma secretase inhibitor, oppositionally indicating an Aβ-mediated mitochondrial toxicity. Analyzing Aβ localization revealed that intracellular levels of Aβ and an increased spatial association of APP/Aβ with mitochondria are associated with reduced mitochondrial respiration. Thus, our data provide marked evidence for a prominent role of intracellular Aβ accumulation in Alzheimer´s disease associated mitochondrial dysfunction. Thereby it highlights the importance of the localization of APP processing and intracellular transport as a decisive factor for mitochondrial function, linking two prominent hallmarks of neurodegenerative diseases.

  14. Metabolic Characterization of Intact Cells Reveals Intracellular Amyloid Beta but Not Its Precursor Protein to Reduce Mitochondrial Respiration

    PubMed Central

    Schaefer, Patrick M.; von Einem, Bjoern; Walther, Paul; Calzia, Enrico; von Arnim, Christine A. F.

    2016-01-01

    One hallmark of Alzheimer´s disease are senile plaques consisting of amyloid beta (Aβ), which derives from the processing of the amyloid precursor protein (APP). Mitochondrial dysfunction has been linked to the pathogenesis of Alzheimer´s disease and both Aβ and APP have been reported to affect mitochondrial function in isolated systems. However, in intact cells, considering a physiological localization of APP and Aβ, it is pending what triggers the mitochondrial defect. Thus, the aim of this study was to dissect the impact of APP versus Aβ in inducing mitochondrial alterations with respect to their subcellular localization. We performed an overexpression of APP or beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), increasing APP and Aβ levels or Aβ alone, respectively. Conducting a comprehensive metabolic characterization we demonstrate that only APP overexpression reduced mitochondrial respiration, despite lower extracellular Aβ levels compared to BACE overexpression. Surprisingly, this could be rescued by a gamma secretase inhibitor, oppositionally indicating an Aβ-mediated mitochondrial toxicity. Analyzing Aβ localization revealed that intracellular levels of Aβ and an increased spatial association of APP/Aβ with mitochondria are associated with reduced mitochondrial respiration. Thus, our data provide marked evidence for a prominent role of intracellular Aβ accumulation in Alzheimer´s disease associated mitochondrial dysfunction. Thereby it highlights the importance of the localization of APP processing and intracellular transport as a decisive factor for mitochondrial function, linking two prominent hallmarks of neurodegenerative diseases. PMID:28005987

  15. The nerve growth factor administrated as eye drops activates mature and precursor cells in subventricular zone of adult rats.

    PubMed

    Tirassa, Paola

    2011-06-01

    The possibility to take advantage from the nerve growth factor (NGF) ability to induce recovery of damaged tissue has been largely explored in animal models and humans. Recently, the successful use of the ocular administration of NGF in ophthalmology, and the evidences that from the eyes NGF can access to the brain have stimulated new fields of research and open further perspectives to the clinical application of this neurotrophin. In our previous studies we have demonstrated the efficacy of NGF eye drop treatment to improved behavioural deficits and recover structural and biochemical alterations occurring follow brain lesion in animals. Since NGF exerts neuroreparative effects in brain by acting on mature neurons and neuronal precursors localised in germinal subventricular zone (SVZ), the present study has been aimed to evaluate the effects of NGF eye drop administration on the expression of the mitotic marker Ki67 in brain of adult rats. We found that a single ocular administration (10 μl) of 200 μg/mL NGF solution is sufficient to enhance the distribution of Ki67 positive cells also expressing p75 neurotrophin receptors in the proliferating layer of the SVZ. In addition, NGF treatment induces an increase of levels of brain derived neurotrophic factor (BDNF) in forebrain. This data further supports the efficacy of ocular applied NGF to affect brain activities and suggests that NGF also by inducing local factors, including BDNF, can activate the machinery regulating the proliferation and maturation of neuronal precursor in brain.

  16. MECHANICAL VIBRATION INHIBITS OSTEOCLAST FORMATION BY REDUCING DC-STAMP RECEPTOR EXPRESSION IN OSTEOCLAST PRECURSOR CELLS

    PubMed Central

    Kulkarni, R.N.; Voglewede, P.A.; Liu, D.

    2014-01-01

    It is well known that physical inactivity leads to loss of muscle mass, but it also causes bone loss. Mechanistically, osteoclastogenesis and bone resorption have recently been shown to be regulated by vibration. However, the underlying mechanism behind the inhibition of osteoclast formation is yet unknown. Therefore, we investigated whether mechanical vibration of osteoclast precursor cells affects osteoclast formation by the involvement of fusion-related molecules such as dendritic cell-specific transmembrane protein (DC-STAMP), and P2X7 receptor (P2X7R). RAW264.7 (a murine osteoclastic-like cell line) cells were treated with 20 ng/ml receptor activator of NF-κB ligand (RANKL). For 3 consecutive days, the cells were subjected to 1 hour of mechanical vibration with 20 µm displacement at a frequency of 4 Hz and compared to the control cells that were treated under the same condition but without the vibration. After 5 days of culture, osteoclast formation was determined. Gene expression of DC-STAMP and P2X7R by RAW264.7 cells were determined after 1 hour mechanical vibration, while protein production of the DC-STAMP was determined after 6 hours of post incubation after vibration. As a result, mechanical vibration of RAW264.7 cells inhibited the formation of osteoclasts. Vibration down-regulated DC-STAMP gene expression by 1.6-fold in the presence of RANKL and by 1.4-fold in the absence of RANKL. Additionally, DC-STAMP protein production was also down-regulated by 1.4-fold in the presence of RANKL and by 1.2-fold in the absence of RANKL in RAW264.7 cells in response to mechanical vibration. However, vibration did not affect P2X7R gene expression. Mouse anti-DC-STAMP antibody inhibited osteoclast formation in the absence of vibration. Our results suggest that mechanical vibration of osteoclast precursor cells reduce DC-STAMP expression in osteoclast precursor cells leading to the inhibition of osteoclast formation. PMID:23994170

  17. Mechanical vibration inhibits osteoclast formation by reducing DC-STAMP receptor expression in osteoclast precursor cells.

    PubMed

    Kulkarni, Rishikesh N; Voglewede, Philip A; Liu, Dawei

    2013-12-01

    It is well known that physical inactivity leads to loss of muscle mass, but it also causes bone loss. Mechanistically, osteoclastogenesis and bone resorption have recently been shown to be regulated by vibration. However, the underlying mechanism behind the inhibition of osteoclast formation is yet unknown. Therefore, we investigated whether mechanical vibration of osteoclast precursor cells affects osteoclast formation by the involvement of fusion-related molecules such as dendritic cell-specific transmembrane protein (DC-STAMP) and P2X7 receptor (P2X7R). RAW264.7 (a murine osteoclastic-like cell line) cells were treated with 20ng/ml receptor activator of NF-κB ligand (RANKL). For 3 consecutive days, the cells were subjected to 1h of mechanical vibration with 20μm displacement at a frequency of 4Hz and compared to the control cells that were treated under the same condition but without the vibration. After 5days of culture, osteoclast formation was determined. Gene expression of DC-STAMP and P2X7R by RAW264.7 cells was determined after 1h of mechanical vibration, while protein production of the DC-STAMP was determined after 6h of postincubation after vibration. As a result, mechanical vibration of RAW264.7 cells inhibited the formation of osteoclasts. Vibration down-regulated DC-STAMP gene expression by 1.6-fold in the presence of RANKL and by 1.4-fold in the absence of RANKL. Additionally, DC-STAMP protein production was also down-regulated by 1.4-fold in the presence of RANKL and by 1.2-fold in the absence of RANKL in RAW264.7 cells in response to mechanical vibration. However, vibration did not affect P2X7R gene expression. Mouse anti-DC-STAMP antibody inhibited osteoclast formation in the absence of vibration. Our results suggest that mechanical vibration of osteoclast precursor cells reduces DC-STAMP expression in osteoclast precursor cells leading to the inhibition of osteoclast formation. © 2013 Elsevier Inc. All rights reserved.

  18. Haematopoietic stem cell induction by somite-derived endothelial cells controlled by meox1.

    PubMed

    Nguyen, Phong Dang; Hollway, Georgina Elizabeth; Sonntag, Carmen; Miles, Lee Barry; Hall, Thomas Edward; Berger, Silke; Fernandez, Kristine Joy; Gurevich, David Baruch; Cole, Nicholas James; Alaei, Sara; Ramialison, Mirana; Sutherland, Robert Lyndsay; Polo, Jose Maria; Lieschke, Graham John; Currie, Peter David

    2014-08-21

    Haematopoietic stem cells (HSCs) are self-renewing stem cells capable of replenishing all blood lineages. In all vertebrate embryos that have been studied, definitive HSCs are generated initially within the dorsal aorta (DA) of the embryonic vasculature by a series of poorly understood inductive events. Previous studies have identified that signalling relayed from adjacent somites coordinates HSC induction, but the nature of this signal has remained elusive. Here we reveal that somite specification of HSCs occurs via the deployment of a specific endothelial precursor population, which arises within a sub-compartment of the zebrafish somite that we have defined as the endotome. Endothelial cells of the endotome are specified within the nascent somite by the activity of the homeobox gene meox1. Specified endotomal cells consequently migrate and colonize the DA, where they induce HSC formation through the deployment of chemokine signalling activated in these cells during endotome formation. Loss of meox1 activity expands the endotome at the expense of a second somitic cell type, the muscle precursors of the dermomyotomal equivalent in zebrafish, the external cell layer. The resulting increase in endotome-derived cells that migrate to colonize the DA generates a dramatic increase in chemokine-dependent HSC induction. This study reveals the molecular basis for a novel somite lineage restriction mechanism and defines a new paradigm in induction of definitive HSCs.

  19. beta-Amyloid1-40 increases expression of beta-amyloid precursor protein in neuronal hybrid cells.

    PubMed

    Le, W; Xie, W J; Nyormoi, O; Ho, B K; Smith, R G; Appel, S H

    1995-11-01

    Studies of cell injury and death in Alzheimer's disease have suggested a prominent role for beta-amyloid peptide (beta-AP), a 40-43-amino-acid peptide derived from a larger membrane glycoprotein, beta-amyloid precursor protein (beta-APP). Previous experiments have demonstrated that beta-AP induces cytotoxicity in a neuronal hybrid cell line (MES 23.5) in vitro. Here, we demonstrate that beta-APP mRNA content is increased 3.5-fold in 24 h after treatment with beta-AP1-40. Accompanying beta-AP1-40-induced cell injury, levels of cell-associated beta-APP and a C-terminal intermediate fragment are increased up to 15-fold, and levels of secreted forms of beta-APP and 12- and 4-kDa fragments are also increased. Application of beta-APP antisense oligodeoxynucleotide reduces both cytotoxicity and beta-APP expression. 6-Hydroxydopamine application or glucose deprivation causes extensive cell damage, but they do not increase beta-APP expression. These results suggest a selective positive feedback mechanism whereby beta-AP may induce cytotoxicity and increase levels of potentially neurotrophic as well as amyloidogenic fragments of beta-APP with the net consequence of further neuronal damage.

  20. Malignant Precursor Cells Pre-Exist in Human Breast DCIS and Require Autophagy for Survival

    PubMed Central

    Espina, Virginia; Mariani, Brian D.; Gallagher, Rosa I.; Tran, Khoa; Banks, Stacey; Wiedemann, Joy; Huryk, Heather; Mueller, Claudius; Adamo, Luana; Deng, Jianghong; Petricoin, Emanuel F.; Pastore, Lucia; Zaman, Syed; Menezes, Geetha; Mize, James; Johal, Jasbir; Edmiston, Kirsten; Liotta, Lance A.

    2010-01-01

    Background While it is accepted that a majority of invasive breast cancer progresses from a ductal carcinoma in situ (DCIS) precursor stage, very little is known about the factors that promote survival of DCIS neoplastic cells within the hypoxic, nutrient deprived intraductal microenvironment. Methodology and Principal Findings We examined the hypothesis that fresh human DCIS lesions contain pre-existing carcinoma precursor cells. We characterized these cells by full genome molecular cytogenetics (Illumina HumanCytoSNP profile), and signal pathway profiling (Reverse Phase Protein Microarray, 59 endpoints), and demonstrated that autophagy is required for survival and anchorage independent growth of the cytogenetically abnormal tumorigenic DCIS cells. Ex vivo organoid culture of fresh human DCIS lesions, without enzymatic treatment or sorting, induced the emergence of neoplastic epithelial cells exhibiting the following characteristics: a) spontaneous generation of hundreds of spheroids and duct-like 3-D structures in culture within 2–4 weeks; b) tumorigenicity in NOD/SCID mice; c) cytogenetically abnormal (copy number loss or gain in chromosomes including 1, 5, 6, 8, 13, 17) compared to the normal karyotype of the non-neoplastic cells in the source patient's breast tissue; d) in vitro migration and invasion of autologous breast stroma; and e) up-regulation of signal pathways linked to, and components of, cellular autophagy. Multiple autophagy markers were present in the patient's original DCIS lesion and the mouse xenograft. We tested whether autophagy was necessary for survival of cytogenetically abnormal DCIS cells. The lysosomotropic inhibitor (chloroquine phosphate) of autophagy completely suppressed the generation of DCIS spheroids/3-D structures, suppressed ex vivo invasion of autologous stroma, induced apoptosis, suppressed autophagy associated proteins including Atg5, AKT/PI3 Kinase and mTOR, eliminated cytogenetically abnormal spheroid forming cells from