WorldWideScience

Sample records for embryonic schwann cell

  1. Biology of Schwann cells.

    Science.gov (United States)

    Kidd, Grahame J; Ohno, Nobuhiko; Trapp, Bruce D

    2013-01-01

    The fundamental roles of Schwann cells during peripheral nerve formation and regeneration have been recognized for more than 100 years, but the cellular and molecular mechanisms that integrate Schwann cell and axonal functions continue to be elucidated. Derived from the embryonic neural crest, Schwann cells differentiate into myelinating cells or bundle multiple unmyelinated axons into Remak fibers. Axons dictate which differentiation path Schwann cells follow, and recent studies have established that axonal neuregulin1 signaling via ErbB2/B3 receptors on Schwann cells is essential for Schwann cell myelination. Extracellular matrix production and interactions mediated by specific integrin and dystroglycan complexes are also critical requisites for Schwann cell-axon interactions. Myelination entails expansion and specialization of the Schwann cell plasma membrane over millimeter distances. Many of the myelin-specific proteins have been identified, and transgenic manipulation of myelin genes have provided novel insights into myelin protein function, including maintenance of axonal integrity and survival. Cellular events that facilitate myelination, including microtubule-based protein and mRNA targeting, and actin based locomotion, have also begun to be understood. Arguably, the most remarkable facet of Schwann cell biology, however, is their vigorous response to axonal damage. Degradation of myelin, dedifferentiation, division, production of axonotrophic factors, and remyelination all underpin the substantial regenerative capacity of the Schwann cells and peripheral nerves. Many of these properties are not shared by CNS fibers, which are myelinated by oligodendrocytes. Dissecting the molecular mechanisms responsible for the complex biology of Schwann cells continues to have practical benefits in identifying novel therapeutic targets not only for Schwann cell-specific diseases but other disorders in which axons degenerate. Copyright © 2013 Elsevier B.V. All rights

  2. Mechanosensitivity of Embryonic Neurites Promotes Their Directional Extension and Schwann Cells Progenitors Migration

    Directory of Open Access Journals (Sweden)

    Gonzalo Rosso

    2017-11-01

    Full Text Available Background/Aims: Migration of Schwann cells (SCs progenitors and neurite outgrowth from embryonic dorsal root ganglions (DRGs are two central events during the development of the peripheral nervous system (PNS. How these two enthralling events preceding myelination are promoted is of great relevance from basic research and clinical aspects alike. Recent evidence demonstrates that biophysical cues (extracellular matrix stiffness and biochemical signaling act in concert to regulate PNS myelination. Microenvironment stiffness of SCs progenitors and embryonic neurites dynamically changes during development. Methods: DRG explants were isolated from day 12.5 to 13.5 mice embryos and plated on laminin-coated substrates with varied stiffness values. After 4 days in culture and immunostaining with specific markers, neurite outgrowth pattern, SCs progenitors migration, and growth cone shape and advance were analyzed with confocal fluorescence microscopy. Results: We found out that growing substrate stiffness promotes directional neurite outgrowth, SCs progenitors migration, growth cone advance and presumably axons fasciculation. Conclusions: DRG explants are in vitro models for the research of PNS development, myelination and regeneration. Consequently, we conclude the following: Our observations point out the importance of mechanosensitivity for the PNS. At the same time, they prompt the investigation of the important yet unclear links between PNS biomechanics and inherited neuropathies with myelination disorders such as Charcot-Marie-Tooth 1A and hereditary neuropathy with liability to pressure palsies. Finally, they encourage the consideration of mechanosensitivity in bioengineering of scaffolds to aid nerve regeneration after injury.

  3. La célula de Schwann The Schwann Cell

    Directory of Open Access Journals (Sweden)

    Spinel Clara

    2004-12-01

    Full Text Available Las neuronas son las células del sistema nervioso y están recubiertas y protegidas por células gliales. En el sistema nerviosos periférico las células de Schwann (CS son la glía de los nervios. Las prolongaciones o neuritas (axón y dendrita de los cuerpos de las neuronas son recubiertas por las CS y constituyen las fibras nerviosas. La relación íntima entre la CS y la neurita se determina durante el desarrollo embrionario. La CS es esencial en la migración correcta de las neuritas hacia su destino final, pero a su vez las neuritas inducen la maduración de las CS. Esta interacción entre CS y neurita está determinada por factores paracrinos y receptores de membrana de las dos células que interactúan, induciendo la diferenciación de las CS en mielinizadas o no, determinando el número de CS necesarias para cubrir las neuritas, formación adecuada de la vaina de mielina y relación correcta de la CS con la matriz extracelular. De esta manera se forma la fibra nerviosa que culmina inervando o recibiendo estímulos en la periferia del cuerpo. Las CS también son la clave de la regeneración de las neuritas en caso de daño de un nervio periférico.The neurones are the cells of the nervous system and are surrounded and protected by glials cells. In the peripheral nervous system the Schwann cells (SC are the glia of the nerves. The prolongations or neurites (axon and dendrite of the neurones bodies are surrounded by the SC to form nervous fibers. The intimate relationship between the SC and the neurite is determined during embryonic development. The SC is essential for the correct migration of the neurites towards their final destination, but the neurites induce SC maturation as well. This interaction between SC and neurite is determined by paracrine factors and membrane receptors of the two interacting cells, inducing the differentiation of SC in myelinic and non-myelinic, determining the correct number of SC necessary to surround the

  4. Schwann cell hamartoma: case report

    Directory of Open Access Journals (Sweden)

    Bulfamante Gaetano

    2011-06-01

    Full Text Available Abstract Background Colorectal polyps of mesenchymal origin represent a small percentage of gastrointestinal (GI lesions. Nevertheless, they are encountered with increasing frequency since the widespread adoption of colonoscopy screening. Case presentation We report a case of a small colonic polyp that presented as intramucosal diffuse spindle cell proliferation with a benign cytological appearance, strong and diffuse immunoreactivity for S-100 protein, and pure Schwann cell phenotype. Careful morphological, immunohistochemical and clinical evaluation emphasize the differences from other stromal colonic lesions and distinguish it from schwannoma, a circumscribed benign nerve sheath tumor that rarely arises in the GI tract. Conclusion As recently proposed, this lesion was finally described as mucosal Schwann cell hamartoma.

  5. Pluripotent Stem Cells for Schwann Cell Engineering

    NARCIS (Netherlands)

    Ma, Ming-San; Boddeke, Erik; Copray, Sjef

    Tissue engineering of Schwann cells (SCs) can serve a number of purposes, such as in vitro SC-related disease modeling, treatment of peripheral nerve diseases or peripheral nerve injury, and, potentially, treatment of CNS diseases. SCs can be generated from autologous stem cells in vitro by

  6. Schwann cell myelination requires Dynein function

    Directory of Open Access Journals (Sweden)

    Langworthy Melissa M

    2012-11-01

    Full Text Available Abstract Background Interaction of Schwann cells with axons triggers signal transduction that drives expression of Pou3f1 and Egr2 transcription factors, which in turn promote myelination. Signal transduction appears to be mediated, at least in part, by cyclic adenosine monophosphate (cAMP because elevation of cAMP levels can stimulate myelination in the absence of axon contact. The mechanisms by which the myelinating signal is conveyed remain unclear. Results By analyzing mutations that disrupt myelination in zebrafish, we learned that Dynein cytoplasmic 1 heavy chain 1 (Dync1h1, which functions as a motor for intracellular molecular trafficking, is required for peripheral myelination. In dync1h1 mutants, Schwann cell progenitors migrated to peripheral nerves but then failed to express Pou3f1 and Egr2 or make myelin membrane. Genetic mosaic experiments revealed that robust Myelin Basic Protein expression required Dync1h1 function within both Schwann cells and axons. Finally, treatment of dync1h1 mutants with a drug to elevate cAMP levels stimulated myelin gene expression. Conclusion Dync1h1 is required for retrograde transport in axons and mutations of Dync1h1 have been implicated in axon disease. Our data now provide evidence that Dync1h1 is also required for efficient myelination of peripheral axons by Schwann cells, perhaps by facilitating signal transduction necessary for myelination.

  7. How Schwann Cells Sort Axons: New Concepts.

    Science.gov (United States)

    Feltri, M Laura; Poitelon, Yannick; Previtali, Stefano Carlo

    2016-06-01

    Peripheral nerves contain large myelinated and small unmyelinated (Remak) fibers that perform different functions. The choice to myelinate or not is dictated to Schwann cells by the axon itself, based on the amount of neuregulin I-type III exposed on its membrane. Peripheral axons are more important in determining the final myelination fate than central axons, and the implications for this difference in Schwann cells and oligodendrocytes are discussed. Interestingly, this choice is reversible during pathology, accounting for the remarkable plasticity of Schwann cells, and contributing to the regenerative potential of the peripheral nervous system. Radial sorting is the process by which Schwann cells choose larger axons to myelinate during development. This crucial morphogenetic step is a prerequisite for myelination and for differentiation of Remak fibers, and is arrested in human diseases due to mutations in genes coding for extracellular matrix and linkage molecules. In this review we will summarize progresses made in the last years by a flurry of reverse genetic experiments in mice and fish. This work revealed novel molecules that control radial sorting, and contributed unexpected ideas to our understanding of the cellular and molecular mechanisms that control radial sorting of axons. © The Author(s) 2015.

  8. Myelinating cocultures of rodent stem cell line-derived neurons and immortalized Schwann cells.

    Science.gov (United States)

    Ishii, Tomohiro; Kawakami, Emiko; Endo, Kentaro; Misawa, Hidemi; Watabe, Kazuhiko

    2017-10-01

    Myelination is one of the most remarkable biological events in the neuron-glia interactions for the development of the mammalian nervous system. To elucidate molecular mechanisms of cell-to-cell interactions in myelin synthesis in vitro, establishment of the myelinating system in cocultures of continuous neuronal and glial cell lines are desirable. In the present study, we performed co-culture experiments using rat neural stem cell-derived neurons or mouse embryonic stem (ES) cell-derived motoneurons with immortalized rat IFRS1 Schwann cells to establish myelinating cultures between these cell lines. Differentiated neurons derived from an adult rat neural stem cell line 1464R or motoneurons derived from a mouse ES cell line NCH4.3, were mixed with IFRS1 Schwann cells, plated, and maintained in serum-free F12 medium with B27 supplement, ascorbic acid, and glial cell line-derived neurotrophic factor. Myelin formation was demonstrated by electron microscopy at 4 weeks in cocultures of 1464R-derived neurons or NCH4.3-derived motoneurons with IFRS1 Schwann cells. These in vitro coculture systems utilizing the rodent stable stem and Schwann cell lines can be useful in studies of peripheral nerve development and regeneration. © 2017 Japanese Society of Neuropathology.

  9. Chemical Conversion of Human Fibroblasts into Functional Schwann Cells

    Directory of Open Access Journals (Sweden)

    Eva C. Thoma

    2014-10-01

    Full Text Available Direct transdifferentiation of somatic cells is a promising approach to obtain patient-specific cells for numerous applications. However, conversion across germ-layer borders often requires ectopic gene expression with unpredictable side effects. Here, we present a gene-free approach that allows efficient conversion of human fibroblasts via a transient progenitor stage into Schwann cells, the major glial cell type of peripheral nerves. Using a multikinase inhibitor, we transdifferentiated fibroblasts into transient neural precursors that were subsequently further differentiated into Schwann cells. The resulting induced Schwann cells (iSCs expressed numerous Schwann cell-specific proteins and displayed neurosupportive and myelination capacity in vitro. Thus, we established a strategy to obtain mature Schwann cells from human postnatal fibroblasts under chemically defined conditions without the introduction of ectopic genes.

  10. Spatiotemporal distribution and function of N-cadherin in postnatal Schwann cells: A matter of adhesion?

    DEFF Research Database (Denmark)

    Corell, Mikael; Wicher, Grzegorz; Limbach, Christoph

    2010-01-01

    During embryonic development of the peripheral nervous system (PNS), the adhesion molecule neuronal cadherin (N-cadherin) is expressed by Schwann cell precursors and associated with axonal growth cones. N-cadherin expression levels decrease as precursors differentiate into Schwann cells....... In this study, we investigated the distribution of N-cadherin in the developing postnatal and adult rat peripheral nervous system. N-cadherin was found primarily in ensheathing glia throughout development, concentrated at neuron-glial or glial-glial contacts of the sciatic nerve, dorsal root ganglia (DRG...

  11. Regulation of retinoid receptors by retinoic acid and axonal contact in Schwann cells.

    Directory of Open Access Journals (Sweden)

    Maria-Jesus Latasa

    Full Text Available BACKGROUND: Schwann cells (SCs are the cell type responsible for the formation of the myelin sheath in the peripheral nervous system (PNS. As retinoic acid (RA and other retinoids have a profound effect as regulators of the myelination program, we sought to investigate how their nuclear receptors levels were regulated in this cell type. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, by using Schwann cells primary cultures from neonatal Wistar rat pups, as well as myelinating cocultures of Schwann cells with embryonic rat dorsal root ganglion sensory neurons, we have found that sustained expression of RXR-γ depends on the continuous presence of a labile activator, while axonal contact mimickers produced an increase in RXR-γ mRNA and protein levels, increment that could be prevented by RA. The upregulation by axonal contact mimickers and the transcriptional downregulation by RA were dependent on de novo protein synthesis and did not involve changes in mRNA stability. On the other hand, RAR-β mRNA levels were only slightly modulated by axonal contact mimickers, while RA produced a strong transcriptional upregulation that was independent of de novo protein synthesis without changes in mRNA stability. CONCLUSIONS/SIGNIFICANCE: All together, our results show that retinoid receptors are regulated in a complex manner in Schwann cells, suggesting that they could have a prominent role as regulators of Schwann cell physiology.

  12. Schwann Cell Phenotype Changes in Aging Human Dental Pulp.

    Science.gov (United States)

    Couve, E; Lovera, M; Suzuki, K; Schmachtenberg, O

    2017-10-01

    Schwann cells are glial cells that support axonal development, maintenance, defense, and regeneration in the peripheral nervous system. There is limited knowledge regarding the organization, plasticity, and aging of Schwann cells within the dental pulp in adult permanent teeth. The present study sought to relate changes in the pattern of Schwann cell phenotypes between young and old adult teeth with neuronal, immune, and vascular components of the dental pulp. Schwann cells are shown to form a prominent glial network at the dentin-pulp interface, consisting of nonmyelinating and myelinating phenotypes, forming a multicellular neuroimmune interface in association with nerve fibers and dendritic cells. Schwann cell phenotypes are recognized by the expression of S100, glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), Sox10, GAP43, and p75NTR markers. In young adult teeth, a dense population of nonmyelinating Schwann cells projects processes in close association with sensory nerve terminals through the odontoblast layer, reaching the adjacent predentin/dentin domain. While GAP43 and p75NTR are highly expressed in nonmyelinating Schwann cells from young adult teeth, the presence of these markers declines significantly in old adult teeth. Myelinated axons, identified by MBP expression, are mainly present at the Raschkow plexus and within nerve bundles in the dental pulp, but their density is significantly reduced in old adult versus young adult teeth. These data reveal age-related changes within the glial network of the dental pulp, in association with a reduction of coronal dental pulp innervation in old adult versus young adult teeth. The prominence of Schwann cells as a cellular component at the dentin-pulp interface supports the notion that their association with sensory nerve terminals and immune system components forms part of an integrated multicellular barrier for defense against pathogens and dentin repair.

  13. Glycolaldehyde induces endoplasmic reticulum stress and apoptosis in Schwann cells

    Directory of Open Access Journals (Sweden)

    Keisuke Sato

    2015-01-01

    Full Text Available Schwann cell injury is caused by diabetic neuropathy. The apoptosis of Schwann cells plays a pivotal role in diabetic nerve dysfunction. Glycolaldehyde is a precursor of advanced glycation end products that contribute to the pathogenesis of diabetic neuropathy. In this study, we examined whether glycolaldehyde induces endoplasmic reticulum (ER stress and apoptosis in rat Schwann cells. Schwann cells treated with 500 μM glycolaldehyde showed morphological changes characteristic of apoptosis. Glycolaldehyde activated apoptotic signals, such as caspase-3 and caspase-8. Furthermore, it induced ER stress response involving RNA-dependent protein kinase-like ER kinase (PERK, inositol-requiring ER-to-nucleus signal kinase 1α (IRE1α, and eukaryotic initiation factor 2α (eIF2α. In addition, glycolaldehyde activated CCAAT/enhancer-binding homologous protein (CHOP, an ER stress response factor crucial to executing apoptosis. Knockdown of nuclear factor E2-related factor 2 (Nrf2, which is involved in the promotion of cell survival following ER stress, enhanced glycolaldehyde-induced cytotoxicity, indicating that Nrf2 plays a protective role in the cytotoxicity caused by glycolaldehyde. Taken together, these findings indicate that glycolaldehyde is capable of inducing apoptosis and ER stress in Schwann cells. The ER stress induced by glycolaldehyde may trigger the glycolaldehyde-induced apoptosis in Schwann cells. This study demonstrated for the first time that glycolaldehyde induced ER stress.

  14. Mesenchymal Stem Cells as an Alternative for Schwann Cells in Rat Spinal Cord Injury

    Science.gov (United States)

    Zaminy, Arash; Shokrgozar, Mohammad Ali; Sadeghi, Yousef; Noroozian, Mohsen; Heidari, Mohammad Hassan; Piryaei, Abbas

    2013-01-01

    Background: Spinal cord has a limited capacity to repair; therefore, medical interventions are necessary for treatment of injuries. Transplantation of Schwann cells has shown a great promising result for spinal cord injury (SCI). However, harvesting Schwann cell has been limited due to donor morbidity and limited expansion capacity. Furthermore, accessible sources such as bone marrow stem cells have drawn attentions to themselves. Therefore, this study was designed to evaluate the effect of bone marrow-derived Schwann cell on functional recovery in adult rats after injury. Methods: Mesenchymal stem cells were cultured from adult rats’ bone marrow and induced into Schwann cells in vitro. Differentiation was confirmed by immunocytochemistry and RT-PCR. Next, Schwann cells were seeded into collagen scaffolds and engrafted in 3 mm lateral hemisection defects. For 8 weeks, motor and sensory improvements were assessed by open field locomotor scale, narrow beam, and tail flick tests. Afterwards, lesioned spinal cord was evaluated by conventional histology and immunohistochemistry. Results: In vitro observations showed that differentiated cells had Schwann cell morphology and markers. In this study, we had four groups (n = 10 each): laminectomy, control, scaffold and scaffold + Schwann cells. Locomotor and sensory scores of cell grafted group were significantly better than control and scaffold groups. In histology, axonal regeneration and remyelination were better than control and scaffold groups. Conclusion: This study demonstrates that bone marrow-derived Schwann cells can be considered as a cell source for Schwann cells in SCI treatment. PMID:23748888

  15. Influenza A virus infection of human Schwann cells in vitro.

    Science.gov (United States)

    Levine, Joshua; Buchman, Craig A; Fregien, Nevis

    2003-01-01

    Sudden sensorineural hearing loss, vestibular neuronitis, vocal fold paralysis and Bell's palsy have been associated with a viral etiology, due to the infection of nerve cells. The goal of this research was to ascertain whether Schwann cells can support infection with human influenza A virus and thereby represent a plausible alternative site for virus-host interaction. Viral infection of Schwann cells may lead to secretion of inflammatory mediators, leukocyte recruitment, demyelination and nerve damage. Cultured human Schwann cells were exposed to human influenza A virus. Infection was assayed at various times post-inoculation (0, 24, 48 and 72 h) using light microscopy, immunocytochemistry and influenza A virus-specific reverse transcriptase polymerase chain reaction (RT-PCR). A group of unexposed cells served as controls. Following exposure to the virus, vacuolization, cellular expansion and detachment from the dish were seen as early as 24 h post-inoculation. The exposed cells demonstrated positive immunocytochemical staining for influenza A virus antigen at 24, 48 and 72 h. Using RT-PCR, a sharp rise in influenza A virus-specific mRNA was detected. Human Schwann cells can be infected with human influenza A virus. Further studies will assess the inflammatory response in this model.

  16. Functional Gap Junctions in the Schwann Cell Myelin Sheath

    Science.gov (United States)

    Balice-Gordon, Rita J.; Bone, Linda J.; Scherer, Steven S.

    1998-01-01

    The Schwann cell myelin sheath is a multilamellar structure with distinct structural domains in which different proteins are localized. Intracellular dye injection and video microscopy were used to show that functional gap junctions are present within the myelin sheath that allow small molecules to diffuse between the adaxonal and perinuclear Schwann cell cytoplasm. Gap junctions are localized to periodic interruptions in the compact myelin called Schmidt–Lanterman incisures and to paranodes; these regions contain at least one gap junction protein, connexin32 (Cx32). The radial diffusion of low molecular weight dyes across the myelin sheath was not interrupted in myelinating Schwann cells from cx32-null mice, indicating that other connexins participate in forming gap junctions in these cells. Owing to the unique geometry of myelinating Schwann cells, a gap junction-mediated radial pathway may be essential for rapid diffusion between the adaxonal and perinuclear cytoplasm, since this radial pathway is approximately one million times faster than the circumferential pathway. PMID:9722620

  17. Dual specificity phosphatase 15 regulates Erk activation in Schwann cells.

    Science.gov (United States)

    Rodríguez-Molina, José F; Lopez-Anido, Camila; Ma, Ki H; Zhang, Chongyu; Olson, Tyler; Muth, Katharina N; Weider, Matthias; Svaren, John

    2017-02-01

    Schwann cells and oligodendrocytes are the myelinating cells of the peripheral and central nervous system, respectively. Despite having different myelin components and different transcription factors driving their terminal differentiation there are shared molecular mechanisms between the two. Sox10 is one common transcription factor required for several steps in development of myelinating glia. However, other factors are divergent as Schwann cells need the transcription factor early growth response 2/Krox20 and oligodendrocytes require Myrf. Likewise, some signaling pathways, like the Erk1/2 kinases, are necessary in both cell types for proper myelination. Nonetheless, the molecular mechanisms that control this shared signaling pathway in myelinating cells remain only partially characterized. The hypothesis of this study is that signaling pathways that are similarly regulated in both Schwann cells and oligodendrocytes play central roles in coordinating the differentiation of myelinating glia. To address this hypothesis, we have used genome-wide binding data to identify a relatively small set of genes that are similarly regulated by Sox10 in myelinating glia. We chose one such gene encoding Dual specificity phosphatase 15 (Dusp15) for further analysis in Schwann cell signaling. RNA interference and gene deletion by genome editing in cultured RT4 and primary Schwann cells showed Dusp15 is necessary for full activation of Erk1/2 phosphorylation. In addition, we show that Dusp15 represses expression of several myelin genes, including myelin basic protein. The data shown here support a mechanism by which early growth response 2 activates myelin genes, but also induces a negative feedback loop through Dusp15 to limit over-expression of myelin genes. © 2016 International Society for Neurochemistry.

  18. Transcriptional Regulation Induced by cAMP Elevation in Mouse Schwann Cells

    Directory of Open Access Journals (Sweden)

    Daniela Schmid

    2014-03-01

    Full Text Available In peripheral nerves, Schwann cell development is regulated by a variety of signals. Some of the aspects of Schwann cell differentiation can be reproduced in vitro in response to forskolin, an adenylyl cyclase activator elevating intracellular cAMP levels. Herein, the effect of forskolin treatment was investigated by a comprehensive genome-wide expression study on primary mouse Schwann cell cultures. Additional to myelin-related genes, many so far unconsidered genes were ascertained to be modulated by forskolin. One of the strongest differentially regulated gene transcripts was the transcription factor Olig1 (oligodendrocyte transcription factor 1, whose mRNA expression levels were reduced in treated Schwann cells. Olig1 protein was localized in myelinating and nonmyelinating Schwann cells within the sciatic nerve as well as in primary Schwann cells, proposing it as a novel transcription factor of the Schwann cell lineage. Data analysis further revealed that a number of differentially expressed genes in forskolin-treated Schwann cells were associated with the ECM (extracellular matrix, underlining its importance during Schwann cell differentiation in vitro. Comparison of samples derived from postnatal sciatic nerves and from both treated and untreated Schwann cell cultures showed considerable differences in gene expression between in vivo and in vitro, allowing us to separate Schwann cell autonomous from tissue-related changes. The whole data set of the cell culture microarray study is provided to offer an interactive search tool for genes of interest.

  19. Schwann cells promote neuronal differentiation of bone marrow ...

    African Journals Online (AJOL)

    Administrator

    2011-04-25

    Apr 25, 2011 ... To culture of Schwann cells, sciatic nerves of adult rats were obtained from adult SD rats under aseptic conditions and placed into Hank's balanced salt solution supplemented. The epineurium and connective tissue were removed under microscope, and the sciatic nerves were cut into 2 to 3 mm fragments, ...

  20. Autoimmunoreactivity to Schwann cells in patients with inflammatory neuropathies

    NARCIS (Netherlands)

    Kwa, Marcel S. G.; van Schaik, Ivo N.; de Jonge, Rosalein R.; Brand, Anneke; Kalaydjieva, Luba; van Belzen, Nico; Vermeulen, Marinus; Baas, Frank

    2003-01-01

    Inflammatory demyelinating neuropathies are characterized by a loss of peripheral nerve myelin. Myelin breakdown is thought to result from an autoimmune reaction towards nerve components. Schwann cells play a crucial role in the synthesis and maintenance of peripheral nerve myelin. An immune attack

  1. Edaravone combined with Schwann cell transplantation may repair spinal cord injury in rats

    Directory of Open Access Journals (Sweden)

    Shu-quan Zhang

    2015-01-01

    Full Text Available Edaravone has been shown to delay neuronal apoptosis, thereby improving nerve function and the microenvironment after spinal cord injury. Edaravone can provide a favorable environment for the treatment of spinal cord injury using Schwann cell transplantation. This study used rat models of complete spinal cord transection at T 9. Six hours later, Schwann cells were transplanted in the head and tail ends of the injury site. Simultaneously, edaravone was injected through the caudal vein. Eight weeks later, the PKH-26-labeled Schwann cells had survived and migrated to the center of the spinal cord injury region in rats after combined treatment with edaravone and Schwann cells. Moreover, the number of PKH-26-labeled Schwann cells in the rat spinal cord was more than that in rats undergoing Schwann cell transplantation alone or rats without any treatment. Horseradish peroxidase retrograde tracing revealed that the number of horseradish peroxidase-positive nerve fibers was greater in rats treated with edaravone combined withSchwann cells than in rats with Schwann cell transplantation alone. The results demonstrated that lower extremity motor function and neurophysiological function were better in rats treated with edaravone and Schwann cells than in rats with Schwann cell transplantation only. These data confirmed that Schwann cell transplantation combined with edaravone injection promoted the regeneration of nerve fibers of rats with spinal cord injury and improved neurological function.

  2. Chronic nerve compression induces concurrent apoptosis and proliferation of Schwann cells.

    Science.gov (United States)

    Gupta, Ranjan; Steward, Oswald

    2003-06-23

    Chronic nerve compression (CNC), as in carpal tunnel syndrome, is a common cause of peripheral nerve dysfunction in humans. Previous studies using animal models have demonstrated progressive demyelination and a slowing of nerve conduction velocity. To characterize the Schwann cell response to CNC, we evaluated total Schwann cell number, apoptosis, and proliferation in an animal model of CNC. Design-based stereologic techniques revealed a striking transient increase in Schwann cell number following CNC. Schwann cell number increased sixfold relative to the normal nerve at the site of compression at 1 month and then slowly declined toward control levels. Nevertheless, assays of apoptosis (TUNEL and an antipoly-ADP-ribose polymerase labeling assays) revealed extensive Schwann cell apoptosis at 2 weeks postcompression, which is during the time when Schwann cell number was increasing. Electron microscopic analysis confirmed that these dramatic changes in Schwann cells occurred in the absence of axon degeneration and axonal swelling and before there were any detectable alterations in nerve conduction velocity. Counts of bromodeoxyuridine-labeled Schwann cells revealed that proliferation occurred concurrently with ongoing apoptosis. To define further the possible mitogenic properties of mechanical stimuli on Schwann cells, we used an in-vitro model to deliver shear stress in the form of laminar fluid flow to pure populations of Schwann cells and confirmed that mechanical stimuli induce Schwann cell proliferation. Our findings indicate that chronic nerve compression induces Schwann cell turnover with minimal axonal injury and support the idea that mechanical stimuli have a direct mitogenic effect on Schwann cells. Copyright 2003 Wiley-Liss, Inc.

  3. [Phenotypic plasticity of neural crest-derived melanocytes and Schwann cells].

    Science.gov (United States)

    Dupin, Elisabeth

    2011-01-01

    expression. This review considers the issue of whether neural crest-derived lineages are endowed with some phenotypic plasticity. Emphasis is put on the ability of pigment cells and Schwann cells to dedifferentiate and reprogram their fate in vitro. To address this question, we have studied the clonal progeny of differentiated Schwann cells and melanocytes after their isolation from the sciatic nerve and the back skin of quail embryos, respectively. When stimulated to proliferate in vitro in the presence of endothelin-3, both cell types were able to dedifferentiate and produce alternative neural crest-derived cell lineages. Individual Schwann cells isolated by FACS, using a glial-specific surface marker, gave rise in culture to pigment cells and myofibroblasts/smooth muscle cells. Treatment of the cultures with endothelin-3 was required for Schwann cell conversion into melanocytes, which involved acquisition of multipotency. Moreover, Schwann cell plasticity could also be induced in vivo: following transplantation into the branchial arch of a young chick host embryo, dedifferentiating Schwann cells were able to integrate the forming head structures of the host and, specifically, to contribute smooth muscle cells to the wall of cranial blood vessels. We also analyzed the in vitro behavior of individual pigment cells obtained by microdissection and enzymatic treatment of quail epidermis at embryonic and hatching stages. In single cell cultures treated with endothelin-3, pigment cells strongly proliferated while rapidly dedifferentiating into unpigmented cells, leading to the formation of large colonies that comprised glial cells and myofibroblasts in addition to melanocytes. By serially subcloning these primary colonies, we could efficiently propagate a bipotent glial-melanocytic precursor that is generated in the progeny of the melanocytic founder. These data therefore suggest that pigment cells have the ability to revert back to the state of self-renewing neural crest

  4. Differential regulation of NAB corepressor genes in Schwann cells

    Directory of Open Access Journals (Sweden)

    Sachdev Shrikesh

    2007-12-01

    Full Text Available Abstract Background Myelination of peripheral nerves by Schwann cells requires not only the Egr2/Krox-20 transactivator, but also the NGFI-A/Egr-binding (NAB corepressors, which modulate activity of Egr2. Previous work has shown that axon-dependent expression of Egr2 is mediated by neuregulin stimulation, and NAB corepressors are co-regulated with Egr2 expression in peripheral nerve development. NAB corepressors have also been implicated in macrophage development, cardiac hypertrophy, prostate carcinogenesis, and feedback regulation involved in hindbrain development. Results To test the mechanism of NAB regulation in Schwann cells, transfection assays revealed that both Nab1 and Nab2 promoters are activated by Egr2 expression. Furthermore, direct binding of Egr2 at these promoters was demonstrated in vivo by chromatin immunoprecipitation analysis of myelinating sciatic nerve, and binding of Egr2 to the Nab2 promoter was stimulated by neuregulin in primary Schwann cells. Although Egr2 expression activates the Nab2 promoter more highly than Nab1, we surprisingly found that only Nab1 – but not Nab2 – expression levels were reduced in sciatic nerve from Egr2 null mice. Analysis of the Nab2 promoter showed that it is also activated by ETS proteins (Ets2 and Etv1/ER81 and is bound by Ets2 in vivo. Conclusion Overall, these results indicate that induction of Nab2 expression in Schwann cells involves not only Egr2, but also ETS proteins that are activated by neuregulin stimulation. Although Nab1 and Nab2 play partially redundant roles, regulation of Nab2 expression by ETS factors explains several observations regarding regulation of NAB genes. Finally, these data suggest that NAB proteins are not only feedback inhibitors of Egr2, but rather that co-induction of Egr2 and NAB genes is involved in forming an Egr2/NAB complex that is crucial for regulation of gene expression.

  5. Dynamic regulation of Schwann cell enhancers after peripheral nerve injury.

    Science.gov (United States)

    Hung, Holly A; Sun, Guannan; Keles, Sunduz; Svaren, John

    2015-03-13

    Myelination of the peripheral nervous system is required for axonal function and long term stability. After peripheral nerve injury, Schwann cells transition from axon myelination to a demyelinated state that supports neuronal survival and ultimately remyelination of axons. Reprogramming of gene expression patterns during development and injury responses is shaped by the actions of distal regulatory elements that integrate the actions of multiple transcription factors. We used ChIP-seq to measure changes in histone H3K27 acetylation, a mark of active enhancers, to identify enhancers in myelinating rat peripheral nerve and their dynamics after demyelinating nerve injury. Analysis of injury-induced enhancers identified enriched motifs for c-Jun, a transcription factor required for Schwann cells to support nerve regeneration. We identify a c-Jun-bound enhancer in the gene for Runx2, a transcription factor induced after nerve injury, and we show that Runx2 is required for activation of other induced genes. In contrast, enhancers that lose H3K27ac after nerve injury are enriched for binding sites of the Sox10 and early growth response 2 (Egr2/Krox20) transcription factors, which are critical determinants of Schwann cell differentiation. Egr2 expression is lost after nerve injury, and many Egr2-binding sites lose H3K27ac after nerve injury. However, the majority of Egr2-bound enhancers retain H3K27ac, indicating that other transcription factors maintain active enhancer status after nerve injury. The global epigenomic changes in H3K27ac deposition pinpoint dynamic changes in enhancers that mediate the effects of transcription factors that control Schwann cell myelination and peripheral nervous system responses to nerve injury. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Schwann Cell Precursors from Human Pluripotent Stem Cells as a Potential Therapeutic Target for Myelin Repair

    Directory of Open Access Journals (Sweden)

    Han-Seop Kim

    2017-06-01

    Full Text Available Schwann cells play a crucial role in successful nerve repair and regeneration by supporting both axonal growth and myelination. However, the sources of human Schwann cells are limited both for studies of Schwann cell development and biology and for the development of treatments for Schwann cell-associated diseases. Here, we provide a rapid and scalable method to produce self-renewing Schwann cell precursors (SCPs from human pluripotent stem cells (hPSCs, using combined sequential treatment with inhibitors of the TGF-β and GSK-3 signaling pathways, and with neuregulin-1 for 18 days under chemically defined conditions. Within 1 week, hPSC-derived SCPs could be differentiated into immature Schwann cells that were functionally confirmed by their secretion of neurotrophic factors and their myelination capacity in vitro and in vivo. We propose that hPSC-derived SCPs are a promising, unlimited source of functional Schwann cells for treating demyelination disorders and injuries to the peripheral nervous system.

  7. Schwann Cell Precursors from Human Pluripotent Stem Cells as a Potential Therapeutic Target for Myelin Repair.

    Science.gov (United States)

    Kim, Han-Seop; Lee, Jungwoon; Lee, Da Yong; Kim, Young-Dae; Kim, Jae Yun; Lim, Hyung Jin; Lim, Sungmin; Cho, Yee Sook

    2017-06-06

    Schwann cells play a crucial role in successful nerve repair and regeneration by supporting both axonal growth and myelination. However, the sources of human Schwann cells are limited both for studies of Schwann cell development and biology and for the development of treatments for Schwann cell-associated diseases. Here, we provide a rapid and scalable method to produce self-renewing Schwann cell precursors (SCPs) from human pluripotent stem cells (hPSCs), using combined sequential treatment with inhibitors of the TGF-β and GSK-3 signaling pathways, and with neuregulin-1 for 18 days under chemically defined conditions. Within 1 week, hPSC-derived SCPs could be differentiated into immature Schwann cells that were functionally confirmed by their secretion of neurotrophic factors and their myelination capacity in vitro and in vivo. We propose that hPSC-derived SCPs are a promising, unlimited source of functional Schwann cells for treating demyelination disorders and injuries to the peripheral nervous system. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Extracellular ATP inhibits Schwann cell dedifferentiation and proliferation in an ex vivo model of Wallerian degeneration

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Youn Ho; Lee, Seo Jin [Department of Anatomy, College of Medicine, Kyung Hee University, Heogi-Dong 1, Dongdaemun-Gu, Seoul 130-701 (Korea, Republic of); Jung, Junyang, E-mail: jjung@khu.ac.kr [Department of Anatomy, College of Medicine, Kyung Hee University, Heogi-Dong 1, Dongdaemun-Gu, Seoul 130-701 (Korea, Republic of)

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer ATP-treated sciatic explants shows the decreased expression of p75NGFR. Black-Right-Pointing-Pointer Extracellular ATP inhibits the expression of phospho-ERK1/2. Black-Right-Pointing-Pointer Lysosomal exocytosis is involved in Schwann cell dedifferentiation. Black-Right-Pointing-Pointer Extracellular ATP blocks Schwann cell proliferation in sciatic explants. -- Abstract: After nerve injury, Schwann cells proliferate and revert to a phenotype that supports nerve regeneration. This phenotype-changing process can be viewed as Schwann cell dedifferentiation. Here, we investigated the role of extracellular ATP in Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Using several markers of Schwann cell dedifferentiation and proliferation in sciatic explants, we found that extracellular ATP inhibits Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Furthermore, the blockage of lysosomal exocytosis in ATP-treated sciatic explants is sufficient to induce Schwann cell dedifferentiation. Together, these findings suggest that ATP-induced lysosomal exocytosis may be involved in Schwann cell dedifferentiation.

  9. ErbB expressing Schwann cells control lateral line progenitor cells via non-cell-autonomous regulation of Wnt/β-catenin.

    Science.gov (United States)

    Lush, Mark E; Piotrowski, Tatjana

    2014-03-18

    Proper orchestration of quiescence and activation of progenitor cells is crucial during embryonic development and adult homeostasis. We took advantage of the zebrafish sensory lateral line to define niche-progenitor interactions to understand how integration of diverse signaling pathways spatially and temporally regulates the coordination of these processes. Our previous studies demonstrated that Schwann cells play a crucial role in negatively regulating lateral line progenitor proliferation. Here we demonstrate that ErbB/Neuregulin signaling is not only required for Schwann cell migration but that it plays a continued role in postmigratory Schwann cells. ErbB expressing Schwann cells inhibit lateral line progenitor proliferation and differentiation through non-cell-autonomous inhibition of Wnt/β-catenin signaling. Subsequent activation of Fgf signaling controls sensory organ differentiation, but not progenitor proliferation. In addition to the lateral line, these findings have important implications for understanding how niche-progenitor cells segregate interactions during development, and how they may go wrong in disease states. DOI: http://dx.doi.org/10.7554/eLife.01832.001.

  10. SOX10 transactivates S100B to suppress Schwann cell proliferation and to promote myelination.

    Directory of Open Access Journals (Sweden)

    Sayaka Fujiwara

    Full Text Available Schwann cells are an important cell source for regenerative therapy for neural disorders. We investigated the role of the transcription factor sex determining region Y (SRY-box 10 (SOX10 in the proliferation and myelination of Schwann cells. SOX10 is predominantly expressed in rat sciatic nerve-derived Schwann cells and is induced shortly after birth. Among transcription factors known to be important for the differentiation of Schwann cells, SOX10 potently transactivates the S100B promoter. In cultures of Schwann cells, overexpressing SOX10 dramatically induces S100B expression, while knocking down SOX10 with shRNA suppresses S100B expression. Here, we identify three core response elements of SOX10 in the S100B promoter and intron 1 with a putative SOX motif. Knockdown of either SOX10 or S100B enhances the proliferation of Schwann cells. In addition, using dissociated cultures of dorsal root ganglia, we demonstrate that suppressing S100B with shRNA impairs myelination of Schwann cells. These results suggest that the SOX10-S100B signaling axis critically regulates Schwann cell proliferation and myelination, and therefore is a putative therapeutic target for neuronal disorders.

  11. Porcine embryonic stem cells

    DEFF Research Database (Denmark)

    Hall, Vanessa Jane

    2008-01-01

    The development of porcine embryonic stem cell lines (pESC) has received renewed interest given the advances being made in the production of immunocompatible transgenic pigs. However, difficulties are evident in the production of pESCs in-vitro. This may largely be attributable to differences...

  12. Glial Cells: The Other Cells of the Nervous System-Schwann Cells ...

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 7; Issue 8. Glial Cells: The Other Cells of the Nervous System - Schwann Cells – Regulators of the Periphery. Yasmin Khan Medha S Rajadhyaksha. Series Article Volume 7 Issue 8 August 2002 pp 8-15 ...

  13. Oriented growth of rat Schwann cells on aligned electrospun poly(methyl methacrylate) nanofibers.

    Science.gov (United States)

    Xia, Haijian; Sun, Xiaochuan; Liu, Dan; Zhou, Yudong; Zhong, Dong

    2016-10-15

    Transplanted Schwann cells have the potential to serve as a support for regenerating neurites after spinal cord injury. However, implanted Schwann cells die off rapidly once transplanted partly owing to the absence of a proper matrix support, with a glia scar and a cavity being present instead at the injury site. For this report, we evaluated aligned electrospun poly(methyl methacrylate) nanofibers as a Schwann cell-loading scaffold in vitro. By monitoring the fluorescence of green fluorescence protein-containing Schwann cells cultured on nanofibers, we found that aligned nanofibers provided better support for the cells than did non-aligned nanofibers. The cells elongated along the long axes of the aligned nanofibers and formed longer cell processes than when the substrate was non-aligned nanofibers. By coculturing Schwann cells with dorsal root ganglion neurons, it was also found that Schwann cells and neurites of dorsal root ganglion neurons could share and both elongate along the orientation of aligned nanofibers and thus they had a higher chance of colocalization than cocultured on film and non-aligned fibers, which might be beneficial to the ensuring process of myelination. The results of the study indicate that aligned electrospun nanofibers may serve as a Schwann cell-loading scaffold for future implantation research. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Mechanosensory organ regeneration in zebrafish depends on a population of multipotent progenitor cells kept latent by Schwann cells.

    Science.gov (United States)

    Sánchez, Mario; Ceci, Maria Laura; Gutiérrez, Daniela; Anguita-Salinas, Consuelo; Allende, Miguel L

    2016-04-07

    Regenerating damaged tissue is a complex process, requiring progenitor cells that must be stimulated to undergo proliferation, differentiation and, often, migratory behaviors and morphological changes. Multiple cell types, both resident within the damaged tissue and recruited to the lesion site, have been shown to participate. However, the cellular and molecular mechanisms involved in the activation of progenitor cell proliferation and differentiation after injury, and their regulation by different cells types, are not fully understood. The zebrafish lateral line is a suitable system to study regeneration because most of its components are fully restored after damage. The posterior lateral line (PLL) is a mechanosensory system that develops embryonically and is initially composed of seven to eight neuromasts distributed along the trunk and tail, connected by a continuous stripe of interneuromastic cells (INCs). The INCs remain in a quiescent state owing to the presence of underlying Schwann cells. They become activated during development to form intercalary neuromasts. However, no studies have described if INCs can participate in a regenerative event, for example, after the total loss of a neuromast. We used electroablation in transgenic larvae expressing fluorescent proteins in PLL components to completely ablate single neuromasts in larvae and adult fish. This injury results in discontinuity of the INCs, Schwann cells, and the PLL nerve. In vivo imaging showed that the INCs fill the gap left after the injury and can regenerate a new neuromast in the injury zone. Further, a single INC is able to divide and form all cell types in a regenerated neuromast and, during this process, it transiently expresses the sox2 gene, a neural progenitor cell marker. We demonstrate a critical role for Schwann cells as negative regulators of INC proliferation and neuromast regeneration, and that this inhibitory property is completely dependent on active ErbB signaling. The potential

  15. Embryonic Stem Cell Markers

    Directory of Open Access Journals (Sweden)

    Lan Ma

    2012-05-01

    Full Text Available Embryonic stem cell (ESC markers are molecules specifically expressed in ES cells. Understanding of the functions of these markers is critical for characterization and elucidation for the mechanism of ESC pluripotent maintenance and self-renewal, therefore helping to accelerate the clinical application of ES cells. Unfortunately, different cell types can share single or sometimes multiple markers; thus the main obstacle in the clinical application of ESC is to purify ES cells from other types of cells, especially tumor cells. Currently, the marker-based flow cytometry (FCM technique and magnetic cell sorting (MACS are the most effective cell isolating methods, and a detailed maker list will help to initially identify, as well as isolate ESCs using these methods. In the current review, we discuss a wide range of cell surface and generic molecular markers that are indicative of the undifferentiated ESCs. Other types of molecules, such as lectins and peptides, which bind to ESC via affinity and specificity, are also summarized. In addition, we review several markers that overlap with tumor stem cells (TSCs, which suggest that uncertainty still exists regarding the benefits of using these markers alone or in various combinations when identifying and isolating cells.

  16. cAMP-dependent reorganization of the Cajal bodies and splicing machinery in cultured Schwann cells.

    Science.gov (United States)

    Fernandez, Rosario; Pena, Emma; Navascues, Joaquin; Casafont, Iñigo; Lafarga, Miguel; Berciano, Maria T

    2002-12-01

    It is well established that forskolin-induced elevation of cAMP results in activation of DNA synthesis in Schwann cell cultures. This promitotic response is partially mediated by the Cdk2, which is required for the transition from the G1 to the S phase of the cell cycle. In the present study, we analyze the effects of cAMP elevation in cultured Schwann cells on the transcriptional activity and on the organization of two nuclear compartments involved in pre-mRNA processing: Cajal bodies (CBs) and splicing factor compartments. Our immunofluorescence and quantitative studies show that forskolin treatment induces a 5.6-fold increase in the proportion of S phase Schwann cells, detected by a short pulse (20 min) of BrdU incorporation. This increase in DNA synthesis correlates with an activation of global transcription, as is indicated by the higher nuclear incorporation of BrU in nascent RNA. Forskolin treatment significantly increases the percentage of Schwann cells containing typical CBs, which concentrate spliceosomal snRNPs and the survival motor neuron (SMN) protein. This increase in the number of CBs closely correlates with the activation of transcription. Moreover, the occurrence of CBs is significantly higher in BrdU (+) cells than in BrdU (-) cells, indicating that entry in the S phase promotes the formation of CBs. During the S phase, Schwann cell nuclei display higher Cdk2 nuclear staining and concentrate this kinase in CBs. Forskolin also induces a redistribution of the pre-mRNA splicing factors in Schwann cells. Primary cultures of Schwann cells provide an excellent physiological model to demonstrate that the assembly of CBs is a transcription- and replication-dependent cellular event. Moreover, the S phase accumulation of Cdk2 observed in Schwann cells supports a functional link between CBs and DNA replication, which is mediated by the possible participation of CBs in the regulatory control of histone gene expression. Copyright 2002 Wiley-Liss, Inc.

  17. Behaviour of oligodendrocytes and Schwann cells in an experimental model of toxic demyelination of the central nervous system

    OpenAIRE

    Dominguita Lühers Graça; Eduardo Fernandes Bondan; Luis Antonio Violin Dias Pereira; Cristina Gevehr Fernandes; Paulo César Maiorka

    2001-01-01

    Oligodendrocytes and Schwann cells are engaged in myelin production, maintenance and repairing respectively in the central nervous system (CNS) and the peripheral nervous system (PNS). Whereas oligodendrocytes act only within the CNS, Schwann cells are able to invade the CNS in order to make new myelin sheaths around demyelinated axons. Both cells have some limitations in their activities, i.e. oligodendrocytes are post-mitotic cells and Schwann cells only get into the CNS in the absence of a...

  18. Schwann cell-derived factors support serotoninergic neuron survival and promote neurite outgrowth

    Directory of Open Access Journals (Sweden)

    R Pellitteri

    2009-12-01

    Full Text Available During embryogenesis and the postnatal period, neurons and glia interact in the development and differentiation of specific populations of nerve cells. Both in the peripheral (PNS and in the central nervous system (CNS, glial cells have been shown in various experimental conditions to constitute a favorable substrate for neural adhesion, neural polarity, shape and axonal extension, while numerous soluble molecules secreted by neurons influence the survival and differentiation of the glial cells themselves. The aim of the present work was to investigate the influence of postnatal Schwann cells (SC on embryonic serotoninergic (5-HT neurons of the raphe, in order to study the possible influence of the peripheral glia on the CNS neurons. Cultures of SC from sciatic nerve of postnatal rats and neurons from rat embryonic rhombencephalon were successfully established and cells were immunocytochemically characterized. The number of 5-HT neurons, and the number and length of their branches were quantified in the cultures of 5-HT neurons, in cultures added with Nerve Growth Factor (NGF and Insulin-like Growth Factor I (IGF-I, in co-cultures with SC and in cultures added with conditioned medium obtained from SC cultures. The results indicated that SC have the capacity to promote the survival and growth of 5-HT neurons in culture, and that this activity is mediated by soluble factors. Although the precise nature and mechanism of action of the growth factor or factors produced by SC in the presence of 5-HT neurons was not identified, our results add more data on the possible activity of the peripheral glia in promoting and enhancing the survival and outgrowth of the CNS neurons.

  19. ATP released by injured neurons activates Schwann cells

    Directory of Open Access Journals (Sweden)

    Samuele eNegro

    2016-05-01

    Full Text Available Injured nerve terminals of neuromuscular junctions (NMJs can regenerate. This remarkable and complex response is governed by molecular signals that are exchanged among the cellular components of this synapse: motor axon nerve terminal (MAT, perisynaptic Schwann cells (PSCs, and muscle fibre. The nature of signals that govern MAT regeneration is ill-known. In the present study the spider toxin α-Latrotoxin has been used as tool to investigate the mechanisms underlying peripheral neuroregeneration. Indeed this neurotoxin induces an acute, specific, localized and fully reversible damage of the presynaptic nerve terminal, and its action mimics the cascade of events that leads to nerve terminal degeneration in injured patients and in many neurodegenerative conditions. Here we provide evidence of an early release by degenerating neurons of ATP as alarm messenger, that contributes to the activation of a series of intracellular pathways within SCs that are crucial for nerve regeneration: Ca2+, cAMP, ERK1/2, and CREB. These results contribute to define the cross-talk taking place among degenerating nerve terminals and PSCs, involved in the functional recovery of the NMJ.

  20. Neuronal merlin influences ERBB2 receptor expression on Schwann cells through neuregulin 1 type III signalling.

    Science.gov (United States)

    Schulz, Alexander; Kyselyova, Anna; Baader, Stephan L; Jung, Marie Juliane; Zoch, Ansgar; Mautner, Victor-Felix; Hagel, Christian; Morrison, Helen

    2014-02-01

    Axonal surface proteins encompass a group of heterogeneous molecules, which exert a variety of different functions in the highly interdependent relationship between axons and Schwann cells. We recently revealed that the tumour suppressor protein merlin, mutated in the hereditary tumour syndrome neurofibromatosis type 2, impacts significantly on axon structure maintenance in the peripheral nervous system. We now report on a role of neuronal merlin in the regulation of the axonal surface protein neuregulin 1 important for modulating Schwann cell differentiation and myelination. Specifically, neuregulin 1 type III expression is reduced in sciatic nerve tissue of neuron-specific knockout animals as well as in biopsies from seven patients with neurofibromatosis type 2. In vitro experiments performed on both the P19 neuronal cell line and primary dorsal root ganglion cells demonstrate the influence of merlin on neuregulin 1 type III expression. Moreover, expression of ERBB2, a Schwann cell receptor for neuregulin 1 ligands is increased in nerve tissue of both neuron-specific merlin knockout animals and patients with neurofibromatosis type 2, demonstrating for the first time that axonal merlin indirectly regulates Schwann cell behaviour. Collectively, we have identified that neuronally expressed merlin can influence Schwann cell activity in a cell-extrinsic manner.

  1. Direct Genesis of Functional Rodent and Human Schwann Cells from Skin Mesenchymal Precursors

    Directory of Open Access Journals (Sweden)

    Matthew P. Krause

    2014-07-01

    Full Text Available Recent reports of directed reprogramming have raised questions about the stability of cell lineages. Here, we have addressed this issue, focusing upon skin-derived precursors (SKPs, a dermally derived precursor cell. We show by lineage tracing that murine SKPs from dorsal skin originate from mesenchymal and not neural crest-derived cells. These mesenchymally derived SKPs can, without genetic manipulation, generate functional Schwann cells, a neural crest cell type, and are highly similar at the transcriptional level to Schwann cells isolated from the peripheral nerve. This is not a mouse-specific phenomenon, since human SKPs that are highly similar at the transcriptome level can be made from neural crest-derived facial and mesodermally derived foreskin dermis and the foreskin SKPs can make myelinating Schwann cells. Thus, nonneural crest-derived mesenchymal precursors can differentiate into bona fide peripheral glia in the absence of genetic manipulation, suggesting that developmentally defined lineage boundaries are more flexible than widely thought.

  2. Interleukin-17 impedes Schwann cell-mediated myelination

    Science.gov (United States)

    2014-01-01

    Background Pro-inflammatory cytokines are known to have deleterious effects on Schwann cells (SCs). Interleukin 17 (IL-17) is a potent pro-inflammatory cytokine that exhibits relevant effects during inflammation in the peripheral nervous system (PNS), and IL-17-secreting cells have been reported within the endoneurium in proximity to the SCs. Methods Here, we analyzed the effects of IL-17 on myelination and the immunological properties of SCs. Dorsal root ganglia (DRG) co-cultures containing neurons and SCs from BL6 mice were used to define the impact of IL-17 on myelination and on SC differentiation; primary SCs were analyzed for RNA and protein expression to define the putative immunological alignment of the SCs. Results SCs were found to functionally express the IL-17 receptors A and B. In DRG cultures, stimulation with IL-17 resulted in reduced myelin synthesis, while pro-myelin gene expression was suppressed at the mRNA level. Neuronal outgrowth and SC viability, as well as structural myelin formation, remained unaffected. Co-cultures exhibited SC-relevant pro-inflammatory markers, such as matrix metalloproteinase 9 and SCs significantly increased the expression of the major histocompatibility complex (MHC) I and exhibited a slight, nonsignificant increase in expression of MHCII, and a transporter associated with antigen presentation (TAP) II molecules relevant for antigen processing and presentation. Conclusions IL-17 may act as a myelin-suppressive mediator in the peripheral nerve, directly propagating SC-mediated demyelination, paralleled by an inflammatory alignment of the SCs. Further analyses are warranted to elucidate the role of IL-17 during inflammation in the PNS in vivo, which could be useful in the development of target therapies. PMID:24678820

  3. SncRNA715 Inhibits Schwann Cell Myelin Basic Protein Synthesis.

    Directory of Open Access Journals (Sweden)

    Christina Müller

    Full Text Available Myelin basic proteins (MBP are major constituents of the myelin sheath in the central nervous system (CNS and the peripheral nervous system (PNS. In the CNS Mbp translation occurs locally at the axon-glial contact site in a neuronal activity-dependent manner. Recently we identified the small non-coding RNA 715 (sncRNA715 as a key inhibitor of Mbp translation during transport in oligodendrocytes. Mbp mRNA localization in Schwann cells has been observed, but has not been investigated in much detail. Here we could confirm translational repression of Mbp mRNA in Schwann cells. We show that sncRNA715 is expressed and its levels correlate inversely with MBP in cultured Schwann cells and in the sciatic nerve in vivo. Furthermore we could reduce MBP protein levels in cultured Schwann cells by increasing the levels of the inhibitory sncRNA715. Our findings suggest similarities in sncRNA715-mediated translational repression of Mbp mRNA in oligodendrocytes and Schwann cells.

  4. Hyperbaric oxygen therapy combined with Schwann cell transplantation promotes spinal cord injury recovery

    Directory of Open Access Journals (Sweden)

    Chuan-gang Peng

    2015-01-01

    Full Text Available Schwann cell transplantation and hyperbaric oxygen therapy each promote recovery from spinal cord injury, but it remains unclear whether their combination improves therapeutic results more than monotherapy. To investigate this, we used Schwann cell transplantation via the tail vein, hyperbaric oxygen therapy, or their combination, in rat models of spinal cord contusion injury. The combined treatment was more effective in improving hindlimb motor function than either treatment alone; injured spinal tissue showed a greater number of neurite-like structures in the injured spinal tissue, somatosensory and motor evoked potential latencies were notably shorter, and their amplitudes greater, after combination therapy than after monotherapy. These findings indicate that Schwann cell transplantation combined with hyperbaric oxygen therapy is more effective than either treatment alone in promoting the recovery of spinal cord in rats after injury.

  5. The Comparison of Schwann Cells Transplantation Effect with Autograft Model in Peripheral Nerve Regeneration in Animal Model

    Directory of Open Access Journals (Sweden)

    Sam Zarbakhsh

    2016-11-01

    Full Text Available Background: Transplantation of Schwann cells can facilitate the regeneration of peripheral nerves. The aim of this study was to comparison of Schwann cells transplantation effect with autograft model in peripheral nerve regeneration in animal model. Materials and Methods: 20 male Wistar rats were randomly were divided into 3 groups: control, Schwann cells transplantation and autograft model. In the control group, a 10 mm segment of the left sciatic nerve was removed and a silicone tube replaced into this nerve gap. In the Schwann cells transplantation group, after placing the silicone tube were transplanted into the tubeabout 500,000 Schwann cells. In the autograft model group, 10 mm segment of the left sciatic nerve is removed and it was implanted to the two nerve endings after reversing. 12 weeks after surgery we evaluated the number of axons, the number of blood vessels and the restored myelin sheath thickness. Results: Histological analysis by using one way ANOVA showed that the number of axons and the thickness of myelin sheath in autograft model group was significantly greater than the other groups, and in the Schwann cells transplantation group was significantly greater than the control group. Moreover, the number of restored blood vessels in the Schwann cells transplantation group was significantly greater than the other groups (P<0.05. Conclusion: The results show that Schwann cells transplantation is effective in peripheral nerve regeneration and it may be a good alternative to autograft method.

  6. mTORC1 promotes proliferation of immature Schwann cells and myelin growth of differentiated Schwann cells

    Science.gov (United States)

    Milbrandt, Jeffrey

    2017-01-01

    The myelination of axons in peripheral nerves requires precisely coordinated proliferation and differentiation of Schwann cells (SCs). We found that the activity of the mechanistic target of rapamycin complex 1 (mTORC1), a key signaling hub for the regulation of cellular growth and proliferation, is progressively extinguished as SCs differentiate during nerve development. To study the effects of different levels of sustained mTORC1 hyperactivity in the SC lineage, we disrupted negative regulators of mTORC1, including TSC2 or TSC1, in developing SCs of mutant mice. Surprisingly, the phenotypes ranged from arrested myelination in nerve development to focal hypermyelination in adulthood, depending on the level and timing of mTORC1 hyperactivity. For example, mice lacking TSC2 in developing SCs displayed hyperproliferation of undifferentiated SCs incompatible with normal myelination. However, these defects and myelination could be rescued by pharmacological mTORC1 inhibition. The subsequent reconstitution of SC mTORC1 hyperactivity in adult animals resulted in focal hypermyelination. Together our data suggest a model in which high mTORC1 activity promotes proliferation of immature SCs and antagonizes SC differentiation during nerve development. Down-regulation of mTORC1 activity is required for terminal SC differentiation and subsequent initiation of myelination. In distinction to this developmental role, excessive SC mTORC1 activity stimulates myelin growth, even overgrowth, in adulthood. Thus, our work delineates two distinct functions of mTORC1 in the SC lineage essential for proper nerve development and myelination. Moreover, our studies show that SCs retain their plasticity to myelinate and remodel myelin via mTORC1 throughout life. PMID:28484008

  7. Platelet-rich plasma gel in combination with Schwann cells for repair of sciatic nerve injury☆

    Science.gov (United States)

    Ye, Fagang; Li, Haiyan; Qiao, Guangxi; Chen, Feng; Tao, Hao; Ji, Aiyu; Hu, Yanling

    2012-01-01

    Bone marrow mesenchymal stem cells were isolated from New Zealand white rabbits, culture-expanded and differentiated into Schwann cell-like cells. Autologous platelet-rich plasma and Schwann cell-like cells were mixed in suspension at a density of 1 × 106 cells/mL, prior to introduction into a poly (lactic-co-glycolic acid) conduit. Fabricated tissue-engineered nerves were implanted into rabbits to bridge 10 mm sciatic nerve defects (platelet-rich plasma group). Controls were established using fibrin as the seeding matrix for Schwann cell-like cells at identical density to construct tissue-engineered nerves (fibrin group). Twelve weeks after implantation, toluidine blue staining and scanning electron microscopy were used to demonstrate an increase in the number of regenerating nerve fibers and thickness of the myelin sheath in the platelet-rich plasma group compared with the fibrin group. Fluoro-gold retrograde labeling revealed that the number of Fluoro-gold-positive neurons in the dorsal root ganglion and the spinal cord anterior horn was greater in the platelet-rich plasma group than in the fibrin group. Electrophysiological examination confirmed that compound muscle action potential and nerve conduction velocity were superior in the platelet-rich plasma group compared with the fibrin group. These results indicate that autologous platelet-rich plasma gel can effectively serve as a seeding matrix for Schwann cell-like cells to construct tissue-engineered nerves to promote peripheral nerve regeneration. PMID:25538751

  8. Neural stem cells promote nerve regeneration through IL12-induced Schwann cell differentiation.

    Science.gov (United States)

    Lee, Don-Ching; Chen, Jong-Hang; Hsu, Tai-Yu; Chang, Li-Hsun; Chang, Hsu; Chi, Ya-Hui; Chiu, Ing-Ming

    2017-03-01

    Regeneration of injured peripheral nerves is a slow, complicated process that could be improved by implantation of neural stem cells (NSCs) or nerve conduit. Implantation of NSCs along with conduits promotes the regeneration of damaged nerve, likely because (i) conduit supports and guides axonal growth from one nerve stump to the other, while preventing fibrous tissue ingrowth and retaining neurotrophic factors; and (ii) implanted NSCs differentiate into Schwann cells and maintain a growth factor enriched microenvironment, which promotes nerve regeneration. In this study, we identified IL12p80 (homodimer of IL12p40) in the cell extracts of implanted nerve conduit combined with NSCs by using protein antibody array and Western blotting. Levels of IL12p80 in these conduits are 1.6-fold higher than those in conduits without NSCs. In the sciatic nerve injury mouse model, implantation of NSCs combined with nerve conduit and IL12p80 improves motor recovery and increases the diameter up to 4.5-fold, at the medial site of the regenerated nerve. In vitro study further revealed that IL12p80 stimulates the Schwann cell differentiation of mouse NSCs through the phosphorylation of signal transducer and activator of transcription 3 (Stat3). These results suggest that IL12p80 can trigger Schwann cell differentiation of mouse NSCs through Stat3 phosphorylation and enhance the functional recovery and the diameter of regenerated nerves in a mouse sciatic nerve injury model. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Myosin-Va-dependent cell-to-cell transfer of RNA from Schwann cells to axons.

    Science.gov (United States)

    Sotelo, José R; Canclini, Lucía; Kun, Alejandra; Sotelo-Silveira, José R; Xu, Lei; Wallrabe, Horst; Calliari, Aldo; Rosso, Gonzalo; Cal, Karina; Mercer, John A

    2013-01-01

    To better understand the role of protein synthesis in axons, we have identified the source of a portion of axonal RNA. We show that proximal segments of transected sciatic nerves accumulate newly-synthesized RNA in axons. This RNA is synthesized in Schwann cells because the RNA was labeled in the complete absence of neuronal cell bodies both in vitro and in vivo. We also demonstrate that the transfer is prevented by disruption of actin and that it fails to occur in the absence of myosin-Va. Our results demonstrate cell-to-cell transfer of RNA and identify part of the mechanism required for transfer. The induction of cell-to-cell RNA transfer by injury suggests that interventions following injury or degeneration, particularly gene therapy, may be accomplished by applying them to nearby glial cells (or implanted stem cells) at the site of injury to promote regeneration.

  10. Early regenerative effects of NGF-transduced Schwann cells in peripheral nerve repair

    NARCIS (Netherlands)

    Shakhbazau, A.; Kawasoe, J.; Hoyng, S.A.; Kumar, R.; van Minnen, J.; Verhaagen, J.; Midha, R.

    2012-01-01

    Peripheral nerve injury leads to a rapid and robust increase in the synthesis of neurotrophins which guide and support regenerating axons. To further optimize neurotrophin supply at the earliest stages of regeneration, we over-expressed NGF in Schwann cells (SCs) by transducing these cells with a

  11. A suspended carbon fiber culture to model myelination by human Schwann cells.

    Science.gov (United States)

    Merolli, Antonio; Mao, Yong; Kohn, Joachim

    2017-04-01

    Understanding of myelination/remyelination process is essential to guide tissue engineering for nerve regeneration. In vitro models currently used are limited to cell population studies and cannot easily identify individual cell contribution to the process. We established a novel model to study the contribution of human Schwann cells to the myelination process. The model avoids the presence of neurons in culture; Schwann cells respond solely to the biophysical properties of an artificial axon. The model uses a single carbon fiber suspended in culture media far from the floor of the well. The fiber provides an elongated structure of defined diameter with 360-degree of surface available for human Schwann cells to wrap around. This model enabled us to spatially and temporally track the myelination by individual Schwann cells along the fiber. We observed cell attachment, elongation and wrapping over a period of 9 days. Cells remained alive and expressed Myelin Basic Protein and Myelin Associated Glycoprotein as expected. Natural and artificial molecules, and external physical factors (e.g., p atterned electrical impulses), may be tested with this model as possible regulators of myelination.

  12. Sulfatase-mediated manipulation of the astrocyte-Schwann cell interface

    NARCIS (Netherlands)

    O'Neill, Paul; Lindsay, Susan L; Pantiru, Andreea; Guimond, Scott E; Fagoe, N.D.; Verhaagen, J.; Turnbull, Jeremy E; Riddell, John S; Barnett, Susan C

    2017-01-01

    Schwann cell (SC) transplantation following spinal cord injury (SCI) may have therapeutic potential. Functional recovery is limited however, due to poor SC interactions with host astrocytes and the induction of astrogliosis. Olfactory ensheathing cells (OECs) are closely related to SCs, but intermix

  13. Fabrication of Aligned Carbon Nanotube/Polycaprolactone/Gelatin Nanofibrous Matrices for Schwann Cell Immobilization

    Directory of Open Access Journals (Sweden)

    Shiao-Wen Tsai

    2014-01-01

    Full Text Available In this study, we utilized a mandrel rotating collector consisting of two parallel, electrically conductive pieces of tape to fabricate aligned electrospun polycaprolactone/gelatin (PG and carbon nanotube/polycaprolactone/gelatin (PGC nanofibrous matrices. Furthermore, we examined the biological performance of the PGC nanofibrous and film matrices using an in vitro culture of RT4-D6P2T rat Schwann cells. Using cell adhesion tests, we found that carbon nanotube inhibited Schwann cell attachment on PGC nanofibrous and film matrices. However, the proliferation rates of Schwann cells were higher when they were immobilized on PGC nanofibrous matrices compared to PGC film matrices. Using western blot analysis, we found that NRG1 and P0 protein expression levels were higher for cells immobilized on PGC nanofibrous matrices compared to PG nanofibrous matrices. However, the carbon nanotube inhibited NRG1 and P0 protein expression in cells immobilized on PGC film matrices. Moreover, the NRG1 and P0 protein expression levels were higher for cells immobilized on PGC nanofibrous matrices compared to PGC film matrices. We found that the matrix topography and composition influenced Schwann cell behavior.

  14. Ectopic expression of polysialylated neural cell adhesion molecule in adult macaque Schwann cells promotes their migration and remyelination potential in the central nervous system.

    Science.gov (United States)

    Bachelin, C; Zujovic, V; Buchet, D; Mallet, J; Baron-Van Evercooren, A

    2010-02-01

    Recent findings suggested that inducing neural cell adhesion molecule polysialylation in rodents is a promising strategy for promoting tissue repair in the injured central nervous system. Since autologous grafting of Schwann cells is one potential strategy to promote central nervous system remyelination, it is essential to show that such a strategy can be translated to adult primate Schwann cells and is of interest for myelin diseases. Adult macaque Schwann cells were transduced with a lentiviral vector encoding sialyltransferase, an enzyme responsible for neural cell adhesion molecule polysialylation. In vitro, we found that ectopic expression of polysialylate promoted adult macaque Schwann cell migration and improved their integration among astrocytes in vitro without modifying their antigenic properties as either non-myelinating or pro-myelinating. In addition, forced expression of polysialylate in adult macaque Schwann cells decreased their adhesion with sister cells. To investigate the ability of adult macaque Schwann cells to integrate and migrate in vivo, focally induced demyelination was targeted to the spinal cord dorsal funiculus of nude mice, and both control and sialyltransferase expressing Schwann cells overexpressing green fluorescein protein were grafted remotely from the lesion site. Analysis of the spatio-temporal distribution of the grafted Schwann cells performed in toto and in situ, showed that in both groups, Schwann cells migrated towards the lesion site. However, migration of sialyltransferase expressing Schwann cells was more efficient than that of control Schwann cells, leading to their accelerated recruitment by the lesion. Moreover, ectopic expression of polysialylated neural cell adhesion molecule promoted adult macaque Schwann cell interaction with reactive astrocytes when exiting the graft, and their 'chain-like' migration along the dorsal midline. The accelerated migration of sialyltransferase expressing Schwann cells to the lesion

  15. The POU proteins Brn-2 and Oct-6 share important functions in Schwann cell development.

    NARCIS (Netherlands)

    M.M. Jaegle (Martine); M. Ghazvini (Mehrnaz); W.J. Mandemakers (Wim); M. Piirsoo (Marko); S. Driegen (Siska); F. Levavasseur (Francoise); S. Raghoenath; F.G. Grosveld (Frank); D. Meijer (Daniëlle)

    2003-01-01

    textabstractThe genetic hierarchy that controls myelination of peripheral nerves by Schwann cells includes the POU domain Oct-6/Scip/Tst-1and the zinc-finger Krox-20/Egr2 transcription factors. These pivotal transcription factors act to control the onset of myelination during

  16. Regulation of Schwann cell proliferation in cultured segments of the adult rat sciatic nerve

    DEFF Research Database (Denmark)

    Svenningsen, Åsa Fex; Kanje, M

    1998-01-01

    of Schwann cells. Removal of extracellular Ca2+ by addition of EGTA to the culture medium suppressed [3H] thymidine incorporation as did the calmodulin inhibitor 48/80. The Ca2+ ionophore A23187 increased incorporation. Staurosporin, an inhibitor of protein kinase C (PKC), suppressed [3H] thymidine...

  17. Lentivirus-Mediated RNA Interference Targeting RhoA Slacks the Migration, Proliferation, and Myelin Formation of Schwann Cells.

    Science.gov (United States)

    Wen, Jinkun; Qian, Changhui; Pan, Mengjie; Wang, Xianghai; Li, Yuanyuan; Lu, Yanmeng; Zhou, Zhitao; Yan, Qing; Li, Lixia; Liu, Zhongying; Wu, Wutian; Guo, Jiasong

    2017-03-01

    RhoA, a member of Rho GTPases family, is known to play an important role in remodeling actin cytoskeleton. During the development of the peripheral nervous system (PNS), Schwann cells undergo proliferation, migration, and radial sorting and finally wrap the related axons compactly to form myelin sheath. All these processes involve actin cytoskeletal remodeling. However, the role of RhoA on Schwann cell during development is still unclear. To address this question, we first used a lentiviral vector-mediated short hairpin (sh) RNA targeting RhoA to knock down the expression of RhoA in the cultured Schwann cells in vitro. Effects of RhoA on Schwann cell proliferation and migration were examined by BrdU assay and transwell assay, respectively. Results of the present study indicated that downregulated RhoA expression in cultured Schwann cells significantly slacked the cells' capabilities of migration and proliferation. Then, we investigated the role of RhoA in the developing rat sciatic nerves. Immunohistology and Western blotting showed that RhoA was mainly expressed in Schwann cells in the sciatic nerves and was peaked at 2 weeks postnatal then kept in low level up to 8 weeks. In the subjected rats whose sciatic nerves were microinjected with lentiviral vectors at postnatal 3 days, we found that the lentiviruses mainly transfected Schwann cells, and the RhoA expression in the transfected Schwann cells was significantly knocked down. Four weeks after lentivirus microinjection, immunohistology and transmission electron microscopy illustrated that RhoA knockdown resulted in hypomyelination and significant decrease of the thickness of myelin in the transfected area. Overall data of current study suggested that RhoA plays a critical role in Schwann cell biology and is essential for myelination in developing peripheral nerve.

  18. Neural-Competent Cells of Adult Human Dermis Belong to the Schwann Lineage

    Directory of Open Access Journals (Sweden)

    Usue Etxaniz

    2014-11-01

    Full Text Available Resident neural precursor cells (NPCs have been reported for a number of adult tissues. Understanding their physiological function or, alternatively, their activation after tissue damage or in vitro manipulation remains an unsolved issue. Here, we investigated the source of human dermal NPCs in adult tissue. By following an unbiased, comprehensive approach employing cell-surface marker screening, cell separation, transcriptomic characterization, and in vivo fate analyses, we found that p75NTR+ precursors of human foreskin can be ascribed to the Schwann (CD56+ and perivascular (CD56− cell lineages. Moreover, neural differentiation potential was restricted to the p75NTR+CD56+ Schwann cells and mediated by SOX2 expression levels. Double-positive NPCs were similarly obtained from human cardiospheres, indicating that this phenomenon might be widespread.

  19. Suspension Matrices for Improved Schwann-Cell Survival after Implantation into the Injured Rat Spinal Cord

    Science.gov (United States)

    Patel, Vivek; Joseph, Gravil; Patel, Amit; Patel, Samik; Bustin, Devin; Mawson, David; Tuesta, Luis M.; Puentes, Rocio; Ghosh, Mousumi

    2010-01-01

    Abstract Trauma to the spinal cord produces endogenously irreversible tissue and functional loss, requiring the application of therapeutic approaches to achieve meaningful restoration. Cellular strategies, in particular Schwann-cell implantation, have shown promise in overcoming many of the obstacles facing successful repair of the injured spinal cord. Here, we show that the implantation of Schwann cells as cell suspensions with in-situ gelling laminin:collagen matrices after spinal-cord contusion significantly enhances long-term cell survival but not proliferation, as well as improves graft vascularization and the degree of axonal in-growth over the standard implantation vehicle, minimal media. The use of a matrix to suspend cells prior to implantation should be an important consideration for achieving improved survival and effectiveness of cellular therapies for future clinical application. PMID:20144012

  20. The influence of electrospun fibre size on Schwann cell behaviour and axonal outgrowth

    Energy Technology Data Exchange (ETDEWEB)

    Gnavi, S., E-mail: sara.gnavi@unito.it [Department of Clinical and Biological Sciences, University of Torino, Orbassano 10043 (Italy); Neuroscience Institute of the Cavalieri-Ottolenghi Foundation, University of Torino, Orbassano 10043 (Italy); Fornasari, B.E., E-mail: benedettaelena.fornasari@unito.it [Department of Clinical and Biological Sciences, University of Torino, Orbassano 10043 (Italy); Neuroscience Institute of the Cavalieri-Ottolenghi Foundation, University of Torino, Orbassano 10043 (Italy); Tonda-Turo, C., E-mail: chiara.tondaturo@polito.it [Politecnico di Torino, Department of Mechanical and Aerospace Engineering, Politecnico of Torino, Torino 10100 (Italy); Ciardelli, G., E-mail: gianluca.ciardelli@polito.it [Politecnico di Torino, Department of Mechanical and Aerospace Engineering, Politecnico of Torino, Torino 10100 (Italy); CNR-IPCF UOS, Pisa 56124 (Italy); Zanetti, M., E-mail: marco.zanetti@unito.it [Nanostructured Interfaces and Surfaces, Department of Chemistry, University of Torino, Torino 10100 (Italy); Geuna, S., E-mail: stefano.geuna@unito.it [Department of Clinical and Biological Sciences, University of Torino, Orbassano 10043 (Italy); Neuroscience Institute of the Cavalieri-Ottolenghi Foundation, University of Torino, Orbassano 10043 (Italy); Perroteau, I., E-mail: isabelle.perroteau@unito.it [Department of Clinical and Biological Sciences, University of Torino, Orbassano 10043 (Italy)

    2015-03-01

    Fibrous substrates functioning as temporary extracellular matrices can be prepared easily by electrospinning, yielding fibrous matrices suitable as internal fillers for nerve guidance channels. In this study, gelatin micro- or nano-fibres were prepared by electrospinning by tuning the gelatin concentration and solution flow rate. The effect of gelatin fibre diameter on cell adhesion and proliferation was tested in vitro using explant cultures of Schwann cells (SC) and dorsal root ganglia (DRG). Cell adhesion was assessed by quantifying the cell spreading area, actin cytoskeleton organization and focal adhesion complex formation. Nano-fibres promoted cell spreading and actin cytoskeleton organization, increasing cellular adhesion and the proliferation rate. However, both migration rate and motility, quantified by transwell and time lapse assays respectively, were greater in cells cultured on micro-fibres. Finally, there was more DRG axon outgrowth on micro-fibres. These data suggest that the topography of electrospun gelatin fibres can be adjusted to modulate SC and axon organization and that both nano- and micro-fibres are promising fillers for the design of devices for peripheral nerve repair. - Highlights: • Electrospinning used to produce gelatin nano- and micro-fibre matrices. • Nano-fibre matrices promote Schwann cell organization and increase proliferation rate. • Micro-fibre matrices promote Schwann cell migration. • Micro-fibre matrices promote axonal outgrowth.

  1. Two factor-based reprogramming of rodent and human fibroblasts into Schwann cells

    Science.gov (United States)

    Mazzara, Pietro Giuseppe; Massimino, Luca; Pellegatta, Marta; Ronchi, Giulia; Ricca, Alessandra; Iannielli, Angelo; Giannelli, Serena Gea; Cursi, Marco; Cancellieri, Cinzia; Sessa, Alessandro; Del Carro, Ubaldo; Quattrini, Angelo; Geuna, Stefano; Gritti, Angela; Taveggia, Carla; Broccoli, Vania

    2017-01-01

    Schwann cells (SCs) generate the myelin wrapping of peripheral nerve axons and are promising candidates for cell therapy. However, to date a renewable source of SCs is lacking. In this study, we show the conversion of skin fibroblasts into induced Schwann cells (iSCs) by driving the expression of two transcription factors, Sox10 and Egr2. iSCs resembled primary SCs in global gene expression profiling and PNS identity. In vitro, iSCs wrapped axons generating compact myelin sheaths with regular nodal structures. Conversely, iSCs from Twitcher mice showed a severe loss in their myelinogenic potential, demonstrating that iSCs can be an attractive system for in vitro modelling of PNS diseases. The same two factors were sufficient to convert human fibroblasts into iSCs as defined by distinctive molecular and functional traits. Generating iSCs through direct conversion of somatic cells offers opportunities for in vitro disease modelling and regenerative therapies. PMID:28169300

  2. Development of a Functional Schwann Cell Phenotype from Autologous Porcine Bone Marrow Mononuclear Cells for Nerve Repair

    Directory of Open Access Journals (Sweden)

    Michael J. Rutten

    2012-01-01

    Full Text Available Adult bone marrow mononuclear cells (BM-MNCs are a potential resource for making Schwann cells to repair damaged peripheral nerves. However, many methods of producing Schwann-like cells can be laborious with the cells lacking a functional phenotype. The objective of this study was to develop a simple and rapid method using autologous BM-MNCs to produce a phenotypic and functional Schwann-like cell. Adult porcine bone marrow was collected and enriched for BM-MNCs using a SEPAX device, then cells cultured in Neurobasal media, 4 mM L-glutamine and 20% serum. After 6–8 days, the cultures expressed Schwann cell markers, S-100, O4, GFAP, were FluoroMyelin positive, but had low p75(NGF expression. Addition of neuregulin (1–25 nM increased p75(NGF levels at 24–48 hrs. We found ATP dose-dependently increased intracellular calcium [Ca2+]i, with nucleotide potency being UTP=ATP>ADP>AMP>adenosine. Suramin blocked the ATP-induced [Ca2+]i but α, β,-methylene-ATP had little effect suggesting an ATP purinergic P2Y2 G-protein-coupled receptor is present. Both the Schwann cell markers and ATP-induced [Ca2+]i sensitivity decreased in cells passaged >20 times. Our studies indicate that autologous BM-MNCs can be induced to form a phenotypic and functional Schwann-like cell which could be used for peripheral nerve repair.

  3. Merlin knockdown in human Schwann cells: clues to vestibular schwannoma tumorigenesis.

    Science.gov (United States)

    Ahmad, Zana; Brown, Carrie Maiorana; Patel, Andrew K; Ryan, Allen F; Ongkeko, Rutherford; Doherty, Joni K

    2010-04-01

    To investigate the early events in molecular progression toward schwannoma tumorigenesis, we developed an in vitro model of human Schwann cell tumorigenesis by merlin knockdown. Neurofibromatosis 2 (NF2)-related and sporadic vestibular schwannoma (VS) exhibit loss of functional merlin (schwannomin). After loss of merlin expression in the Schwann cell, the initial steps toward VS tumorigenesis are unknown. Merlin, a putative tumor suppressor protein, interacts with many cellular proteins, regulating their function. Among these are receptor tyrosine kinases, including the epidermal growth factor receptor family B (ErbB) family receptors epidermal growth factor receptor and ErbB2. Functional merlin interacts with and internalizes these growth factor receptors, silencing their proliferation and survival signaling. Deregulation of CD44, the cell adhesion/signaling molecule and cancer stem cell marker, has also been implicated in VS tumorigenesis. Merlin knockdown was performed using small interfering RNA transfection into human Schwann cell primary cultures. Knockdown was confirmed by real-time quantitative PCR, immunofluorescence, and Western analysis. Expression profiles of ErbB, merlin, and the stem cell markers nestin and CD44 were examined in knockdowns. Proliferation rate was assessed with bromodeoxyuridine incorporation, and radiation sensitivity was assessed using the Annexin assay in knockdowns versus controls. Merlin knockdowns demonstrated increased proliferation rate, upregulation of epidermal growth factor receptor, ErbB2, and ErbB3, CD44, and nestin. Short-term merlin depletion had no effect on gamma irradiation sensitivity compared with controls. Merlin depletion results in deregulation of ErbB receptor signaling, promotes a dedifferentiated state, and increases Schwann cell proliferation, suggesting critical steps toward schwannoma tumorigenesis.

  4. MAL Overexpression Leads to Disturbed Expression of Genes That Influence Cytoskeletal Organization and Differentiation of Schwann Cells

    Directory of Open Access Journals (Sweden)

    Daniela Schmid

    2014-09-01

    Full Text Available In the developing peripheral nervous system, a coordinated reciprocal signaling between Schwann cells and axons is crucial for accurate myelination. The myelin and lymphocyte protein MAL is a component of lipid rafts that is important for targeting proteins and lipids to distinct domains. MAL overexpression impedes peripheral myelinogenesis, which is evident by a delayed onset of myelination and reduced expression of the myelin protein zero (Mpz/P0 and the low-affinity neurotrophin receptor p75NTR . This study shows that MAL overexpression leads to a significant reduction of Mpz and p75NTR expression in primary mouse Schwann cell cultures, which was already evident before differentiation, implicating an effect of MAL in early Schwann cell development. Their transcription was robustly reduced, despite normal expression of essential transcription factors and receptors. Further, the cAMP response element-binding protein (CREB and phosphoinositide 3-kinase signaling pathways important for Schwann cell differentiation were correctly induced, highlighting that other so far unknown rate limiting factors do exist. We identified novel genes expressed by Schwann cells in a MAL-dependent manner in vivo and in vitro. A number of those, including S100a4, RhoU and Krt23, are implicated in cytoskeletal organization and plasma membrane dynamics. We showed that S100a4 is predominantly expressed by nonmyelinating Schwann cells, whereas RhoU was localized within myelin membranes, and Krt23 was detected in nonmyelinating as well as in myelinating Schwann cells. Their differential expression during early peripheral nerve development further underlines their possible role in influencing Schwann cell differentiation and myelination.

  5. Schwann cell expression of an oligodendrocyte-like remyelinating pattern after ethidium bromide injection in the rat spinal cord

    OpenAIRE

    Eduardo Fernandes Bondan; Maria Anete Lallo; Maria de Fátima Monteiro Martins; Dominguita Luhers Graça

    2010-01-01

    Schwann cells are recognized by their capacity of producing single internodes of myelin around axons of the peripheral nervous system. In the ethidium bromide (EB) model of primary demyelination in the brainstem, it is observed the entry of Schwann cells into the central nervous system in order to contribute to the myelin repair performed by the oligodendrocytes that survived to the EB gliotoxic action, being able to even remyelinate more than one axon at the same time, in a pattern of repair...

  6. miR-30c promotes Schwann cell remyelination following peripheral nerve injury

    Directory of Open Access Journals (Sweden)

    Sheng Yi

    2017-01-01

    Full Text Available Differential expression of miRNAs occurs in injured proximal nerve stumps and includes miRNAs that are firstly down-regulated and then gradually up-regulated following nerve injury. These miRNAs might be related to a Schwann cell phenotypic switch. miR-30c, as a member of this group, was further investigated in the current study. Sprague-Dawley rats underwent sciatic nerve transection and proximal nerve stumps were collected at 1, 4, 7, 14, 21, and 28 days post injury for analysis. Following sciatic nerve injury, miR-30c was down-regulated, reaching a minimum on day 4, and was then upregulated to normal levels. Schwann cells were isolated from neonatal rat sciatic nerve stumps, then transfected with miR-30c agomir and co-cultured in vitro with dorsal root ganglia. The enhanced expression of miR-30c robustly increased the amount of myelin-associated protein in the co-cultured dorsal root ganglia and Schwann cells. We then modeled sciatic nerve crush injury in vivo in Sprague-Dawley rats and tested the effect of perineural injection of miR-30c agomir on myelin sheath regeneration. Fourteen days after surgery, sciatic nerve stumps were harvested and subjected to immunohistochemistry, western blot analysis, and transmission electron microscopy. The direct injection of miR-30c stimulated the formation of myelin sheath, thus contributing to peripheral nerve regeneration. Overall, our findings indicate that miR-30c can promote Schwann cell myelination following peripheral nerve injury. The functional study of miR-30c will benefit the discovery of new therapeutic targets and the development of new treatment strategies for peripheral nerve regeneration.

  7. Lipoamide Inhibits NF1 Deficiency-induced Epithelial-Mesenchymal Transition in Murine Schwann Cells.

    Science.gov (United States)

    Zhang, Yuchen; Zhou, Rongsheng; Qu, Yiping; Shu, Maoguo; Guo, Shuzhong; Bai, Zhuanli

    2017-11-29

    Neurofibromatosis type I (NF1) is one of the most common neurocutaneous syndromes characterized by development of adult neurofibromas which is mainly made up of Schwann cells. The disease is generally accepted to be caused by inactivation mutation of Nf1 gene. And Nf1 deficiency had been reported to lead to ROS overproduction and epithelial-mesenchymal transition (EMT) phenotype. This study was designed to investigate whether excessive ROS conferred to Nf1 deficiency-induced EMT in Schwann cells. Colony formation, wound healing assay and transwell assay was used to evaluate the effects of stable Nf1 knockdown in SW10 Schwann cells. Western blot and ROS assay was conducted to explore the molecular mechanisms of Nf1 inactivation in tumorigenesis. Animal experiments were performed to assess the inhibitory effects of lipoamide, which is the neutral amide of α-lipoic acid and functions as a potent antioxidant to scavenge ROS, on Nf1-deficiency tumor growth in vivo. Nf1 knockdown enhanced the cellular capacities of proliferation, migration and invasion, promoted ROS generation, decreased the expression of epithelial surface marker E-cadherin, and up-regulated several EMT-associated molecules in Schwann cells. Moreover, lipoamide dose-dependently inhibited not only Nf1 deficiency-induced EMT but also spontaneous EMT. Furthermore, lipoamide markedly suppresses tumor growth in a mouse model of NF1-associated neurofibroma. Our results clearly reveal that ROS overproduction is responsible for Nf1 deficiency-induced EMT and plays a crucial role in NF1 tumor growth. The findings presented herein shed light on the potential of antioxidant therapy to prevent the progression of NF1-associated neurofibroma. Copyright © 2017 IMSS. Published by Elsevier Inc. All rights reserved.

  8. Age-Dependent Schwann Cell Phenotype Regulation Following Peripheral Nerve Injury.

    Science.gov (United States)

    Chen, Wayne A; Luo, T David; Barnwell, Jonathan C; Smith, Thomas L; Li, Zhongyu

    2017-12-01

    Schwann cells are integral to the regenerative capacity of the peripheral nervous system, which declines after adolescence. The mechanisms underlying this decline are poorly understood. This study sought to compare the protein expression of Notch, c-Jun, and Krox-20 after nerve crush injury in adolescent and young adult rats. We hypothesized that these Schwann cell myelinating regulatory factors are down-regulated after nerve injury in an age-dependent fashion. Adolescent (2 months old) and young adult (12 months old) rats (n = 48) underwent sciatic nerve crush injury. Protein expression of Notch, c-Jun, and Krox-20 was quantified by Western blot analysis at 1, 3, and 7 days post-injury. Functional recovery was assessed in a separate group of animals (n = 8) by gait analysis (sciatic functional index) and electromyography (compound motor action potential) over an 8-week post-injury period. Young adult rats demonstrated a trend of delayed onset of the dedifferentiating regulatory factors, Notch and c-Jun, corresponding to the delayed functional recovery observed in young adult rats compared to adolescent rats. Compound motor action potential area was significantly greater in adolescent rats relative to young adult rats, while amplitude and velocity trended toward statistical significance. The process of Schwann cell dedifferentiation following peripheral nerve injury shows different trends with age. These trends of delayed onset of key regulatory factors responsible for Schwann cell myelination may be one of many possible factors mediating the significant differences in functional recovery between adolescent and young adult rats following peripheral nerve injury.

  9. Comparative Analysis of the Cell Fates of Induced Schwann Cells from Subcutaneous Fat Tissue and Naïve Schwann Cells in the Sciatic Nerve Injury Model

    Directory of Open Access Journals (Sweden)

    Mingzi Zhang

    2017-01-01

    Full Text Available Purpose. The fate and function of the induced Schwann cells (iSCs like cells from adipose tissue have not been critically evaluated in vivo after transplantation. The objective of this study is to compare the fate of iSCs with naïve SCs (nSCs after transplantation into the lesion sites of sciatic nerve, respectively. Methods. Adipose-derived stem cells from eGFP-expressing transgenic rat’s subcutaneous fat were induced to iSCs in vitro. iSCs were injected to the sciatic nerve lesion area after crush injury and the cells fate was comparatively analyzed with that of nSCs from the same rat. Results. At 12 weeks after transplantation, nSCs were detected only in the restricted area of cell transplantation site but iSCs were widely distributed all over the sciatic nerve. Based on double fluorescence observations, both iSCs and naïve ones were colocalized with P0-expressing myelin sheath, outbound by laminin-expressing basal membrane, and terminated at contactin-associated protein-expressing doublets. However, some of iSCs were also differentiated to the fibrocyte/fibroblast-like cells. In the histological analysis of repaired sciatic nerves, axon density was higher in iSC-received group than in the nSCs group and normal sciatic nerve. Conclusion. iSCs induced from subcutaneous fat tissues have higher engraftment and migration capacity than nSCs.

  10. Brain-derived neurotrophic factor promotes nerve regeneration by activating the JAK/STAT pathway in Schwann cells.

    Science.gov (United States)

    Lin, Guiting; Zhang, Haiyang; Sun, Fionna; Lu, Zhihua; Reed-Maldonado, Amanda; Lee, Yung-Chin; Wang, Guifang; Banie, Lia; Lue, Tom F

    2016-04-01

    Radical prostatectomy (RP) carries the risk of erectile dysfunction (ED) due to cavernous nerve (CN) injury. Schwann cells are essential for the maintenance of integrity and function of peripheral nerves such as the CNs. We hypothesize that brain-derived neurotrophic factor (BDNF) activates the Janus kinase (JAK)/(signal transducer and activator of transcription) STAT pathway in Schwann cells, not in neuronal axonal fibers, with the resultant secretion of cytokines from Schwann cells to facilitate nerve recovery. Using four different cell lines-human neuroblastoma BE(2)-C and SH-SY5Y, human Schwann cell (HSC), and rat Schwann cell (RSC) RT4-D6P2T-we assessed the effect of BDNF application on the activation of the JAK/STAT pathway. We also assessed the time response of JAK/STAT pathway activation in RSCs and HSCs after BDNF treatment. We then assayed cytokine release from HSCs as a response to BDNF treatment using oncostatin M and IL6 as markers. We showed extensive phosphorylation of STAT3/STAT1 by BDNF at high dose (100 pM) in RSCs, with no JAK/STAT pathway activation in human neuroblastoma cell lines. The time response of JAK/STAT pathway activation in RSCs and HSCs after BDNF treatment showed an initial peak at shortly after treatment and then a second higher peak at 24-48 hours. Cytokine release from HSCs increased progressively after BDNF application, reaching statistical significance for IL6. We demonstrated for the first time the indirect mechanism of BDNF enhancement of nerve regeneration through the activation of JAK/STAT pathway in Schwann cells, rather than directly on neurons. As a result of BDNF application, Schwann cells produce cytokines that promote nerve regeneration.

  11. Schwann Cells Metabolize Extracellular 2′,3′-cAMP to 2′-AMP

    Science.gov (United States)

    Verrier, Jonathan D.; Kochanek, Patrick M.

    2015-01-01

    The 3′,5′-cAMP–adenosine pathway (3′,5′-cAMP→5′-AMP→adenosine) and the 2′,3′-cAMP–adenosine pathway (2′,3′-cAMP→2′-AMP/3′-AMP→adenosine) are active in the brain. Oligodendrocytes participate in the brain 2′,3′-cAMP–adenosine pathway via their robust expression of 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase; converts 2′,3′-cAMP to 2′-AMP). Because Schwann cells also express CNPase, it is conceivable that the 2′,3′-cAMP–adenosine pathway exists in the peripheral nervous system. To test this and to compare the 2′,3′-cAMP–adenosine pathway to the 3′,5′-cAMP–adenosine pathway in Schwann cells, we examined the metabolism of 2′,3′-cAMP, 2′-AMP, 3′-AMP, 3′,5′-cAMP, and 5′-AMP in primary rat Schwann cells in culture. Addition of 2′,3′-cAMP (3, 10, and 30 µM) to Schwann cells increased levels of 2′-AMP in the medium from 0.006 ± 0.002 to 21 ± 2, 70 ± 3, and 187 ± 10 nM/µg protein, respectively; in contrast, Schwann cells had little ability to convert 2′,3′-cAMP to 3′-AMP or 3′,5′-cAMP to either 3′-AMP or 5′-AMP. Although Schwann cells slightly converted 2′,3′-cAMP and 2′-AMP to adenosine, they did so at very modest rates (e.g., 5- and 3-fold, respectively, more slowly compared with our previously reported studies in oligodendrocytes). Using transected myelinated rat sciatic nerves in culture medium, we observed a time-related increase in endogenous intracellular 2′,3′-cAMP and extracellular 2′-AMP. These findings indicate that Schwann cells do not have a robust 3′,5′-cAMP–adenosine pathway but do have a 2′,3′-cAMP–adenosine pathway; however, because the pathway mostly involves 2′-AMP formation rather than 3′-AMP, and because the conversion of 2′-AMP to adenosine is slow, metabolism of 2′,3′-cAMP mostly results in the accumulation of 2′-AMP. Accumulation of 2′-AMP in peripheral nerves postinjury could have

  12. Myelinating Schwann Cell Polarity and Mechanically-Driven Myelin Sheath Elongation.

    Science.gov (United States)

    Tricaud, Nicolas

    2017-01-01

    Myelin sheath geometry, encompassing myelin sheath thickness relative to internodal length, is critical to optimize nerve conduction velocity and these parameters are carefully adjusted by the myelinating cells in mammals. In the central nervous system these adjustments could regulate neuronal activities while in the peripheral nervous system they lead to the optimization and the reliability of the nerve conduction velocity. However, the physiological and cellular mechanisms that underlie myelin sheath geometry regulation are not yet fully elucidated. In peripheral nerves the myelinating Schwann cell uses several molecular mechanisms to reach and maintain the correct myelin sheath geometry, such that myelin sheath thickness and internodal length are regulated independently. One of these mechanisms is the epithelial-like cell polarization process that occurs during the early phases of the myelin biogenesis. Epithelial cell polarization factors are known to control cell size and morphology in invertebrates and mammals making these processes critical in the organogenesis. Correlative data indicate that internodal length is regulated by postnatal body growth that elongates peripheral nerves in mammals. In addition, the mechanical stretching of peripheral nerves in adult animals shows that myelin sheath length can be increased by mechanical cues. Recent results describe the important role of YAP/TAZ co-transcription factors during Schwann cell myelination and their functions have linked to the mechanotransduction through the HIPPO pathway and the epithelial polarity factor Crb3. In this review the molecular mechanisms that govern mechanically-driven myelin sheath elongation and how a Schwann cell can modulate internodal myelin sheath length, independent of internodal thickness, will be discussed regarding these recent data. In addition, the potential relevance of these mechanosensitive mechanisms in peripheral pathologies will be highlighted.

  13. In vitro cytocompatibility assessment of amorphous carbon structures using neuroblastoma and Schwann cells.

    Science.gov (United States)

    Jain, Shilpee; Sharma, Ashutosh; Basu, Bikramjit

    2013-05-01

    The development of scaffolds for neural tissue engineering application requires an understanding of cell adhesion, proliferation, and migration of neuronal cells. Considering the potential application of carbon as scaffold materials and the lack of understanding of compatibility of amorphous carbon with neuronal cells, the carbon-based materials in the forms of carbon films and continuous electrospun carbon nanofibers having average diameter of ~200 nm are being investigated with or without ultraviolet (UV) and oxy-plasma (OP) treatments for cytocompatibility property using mouse Neuroblastoma (N2a) and rat Schwann cells (RT4-D6P2T). The use of Raman spectroscopy in combination with Fourier transform infrared (FTIR) and X-ray diffraction establishes the amorphous nature and surface-bonding characteristics of the studied carbon materials. Although both UV and OP treatments make carbon surfaces more hydrophilic, the cell viability of N2a cells is statistically more significant on OP treated fibers/films compared to UV fiber/film substrates after 4 days in culture. The electrospun carbon fibrous substrate provides the physical guidance to the cultured Schwann cells. Overall, the experimental results of this study demonstrate that the electrospun amorphous carbon nanofibrous scaffolds can be used as a suitable biomaterial substrate for supporting cell adhesion and proliferation of neuronal cells in the context of their applications as artificial nerve implants. Copyright © 2013 Wiley Periodicals, Inc.

  14. Alginate-matrigel microencapsulated schwann cells for inducible secretion of glial cell line derived neurotrophic factor.

    Science.gov (United States)

    de Guzman, Roche C; Ereifej, Evon S; Broadrick, Kristy M; Rogers, Richard A; VandeVord, Pamela J

    2008-10-01

    Controlled expression of glial cell line derived neurotrophic factor (Gdnf) can be integrated in the development of a system for repair of injured peripheral nerves. This delivery strategy was demonstrated via inducible Gdnf from microencapsulated cells in barium alginate. The Schwann cell line RT4-D6P2T was initially modified utilizing an ecdysone-based stable transfection system to produce RT4-Gdnf cells. During construct preparation, it was found that C6 cells (where Gdnf cDNA was isolated) make three Gdnf transcript variants. Additionally, the importance of 5' untranslated region to drive biologically-functional Gdnf synthesis was shown. Encapsulation of RT4-Gdnf in 1% alginate was then performed. It was determined that cells were able to survive at least 1 month in vitro using starting densities of 20, 200 and 2000 cells/capsule and barium ion concentrations of 10, 50, 100 and 200 mM. Most importantly, encapsulated cells secreted exogenous Gdnf upon ponasterone A induction. Mixture of basement membrane extract Matrigel to alginate promoted increased proliferation, cell spreading and Gdnf release. Finally, compression tests showed that cell-loaded microcapsules fractured at 75% diameter compression with 38 kPa of stress. Regulated Gdnf release from these microcapsules in vivo may potentially aid in the regeneration of damaged nerves.

  15. Cooperative interaction of hepatocyte growth factor and neuregulin regulates Schwann cell migration and proliferation through Grb2-associated binder-2 in peripheral nerve repair.

    Science.gov (United States)

    Shin, Yoon Kyoung; Jang, So Young; Yun, Seoug Hoon; Choi, Yun Young; Yoon, Byeol-A; Jo, Young Rae; Park, So Young; Pak, Min Gyoung; Park, Joo In; Park, Hwan Tae

    2017-11-01

    The sequential reactive changes in Schwann cell phenotypes in transected peripheral nerves, including dedifferentiation, proliferation and migration, are essential for nerve repair. Even though the injury-induced migratory and proliferative behaviors of Schwann cells resemble epithelial and mesenchymal transition (EMT) in tumors, the molecular mechanisms underlying this phenotypic change of Schwann cells are still unclear. Here we show that the reactive Schwann cells exhibit migratory features dependent on the expression of a scaffolding oncoprotein Grb2-associated binder-2 (Gab2), which was transcriptionally induced by neuregulin 1-ErbB2 signaling following nerve injury. Injury-induced Gab2 expression was dependent on c-Jun, a transcription factor critical to a Schwann cell reprograming into a repair-type cell. Interestingly, the injury-induced activation (tyrosine phosphorylation) of Gab2 in Schwann cells was regulated by an EMT signal, the hepatocyte growth factor-c-Met signaling, but not by neuregulin 1. Gab2 knockout mice exhibited a deficit in nerve repair after nerve transection due to limited Schwann cell migration. Furthermore, Gab2 was required for the proliferation of Schwann cells following nerve injury and in vitro, and was over-expressed in human Schwann cell-derived tumors. In contrast, the tyrosine phosphorylation of Gab1 after nerve injury was principally regulated by the neuregulin 1-ErbB2 signaling and was indispensable for remyelination after crush injury, but not for the proliferation and migration of Schwann cells. Our findings indicate that Gab1 and Gab2 in Schwann cells are nonredundant and play a crucial role in peripheral nerve repair. © 2017 Wiley Periodicals, Inc.

  16. Myelin repair by Schwann cells in the regenerating goldfish visual pathway: regional patterns revealed by X-irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Nona, S.N.; Stafford, C.A.; Cronly-Dillon, J.R. (Manchester Univ. (United Kingdom). Inst. of Science and Technology); Duncan, A. (Guy' s Hospital, London (United Kingdom). Dept. of Anatomy); Scholes, J. (University Coll., London (United Kingdom))

    1994-07-01

    In the regenerating goldfish optic nerves, Schwann cells of unknown origin reliably infiltrate the lesion site forming a band of peripheral-type myelinating tissue by 1-2 months, sharply demarcated form the adjacent new CNS myelin. To investigate this effect, we have interfered with cell proliferation by locally X-irradiating the fish visual pathway 24 h after the lesion. As assayed by immunohistochemistry and EM, irradiation retards until 6 months formation of new myelin by Schwann cells at the lesion site, and virtually abolishes oligodendrocyte myelination distally, but has little or no effect on nerve fibre regrowth. Optic nerve astrocyte processes normally fail to re-infiltrate the lesion, but re-occupy it after irradiation, suggesting that they are normally excluded by early cell proliferation at this site. Moreover, scattered myelinating Schwann cells also appear in the oligodendrocyte-depleted distal optic nerve after irradiation, although only as far as the optic tract. (Author).

  17. The Scaffolding Protein, Grb2-associated Binder-1, in Skeletal Muscles and Terminal Schwann Cells Regulates Postnatal Neuromuscular Synapse Maturation.

    Science.gov (United States)

    Park, So Young; Jang, So Young; Shin, Yoon Kyoung; Jung, Dong Keun; Yoon, Byeol A; Kim, Jong Kook; Jo, Young Rae; Lee, Hye Jeong; Park, Hwan Tae

    2017-06-01

    The vertebrate neuromuscular junction (NMJ) is considered as a "tripartite synapse" consisting of a motor axon terminal, a muscle endplate, and terminal Schwann cells that envelope the motor axon terminal. The neuregulin 1 (NRG1)-ErbB2 signaling pathway plays an important role in the development of the NMJ. We previously showed that Grb2-associated binder 1 (Gab1), a scaffolding mediator of receptor tyrosine kinase signaling, is required for NRG1-induced peripheral nerve myelination. Here, we determined the role of Gab1 in the development of the NMJ using muscle-specific conditional Gab1 knockout mice. The mutant mice showed delayed postnatal maturation of the NMJ. Furthermore, the selective loss of the gab1 gene in terminal Schwann cells produced delayed synaptic elimination with abnormal morphology of the motor endplate, suggesting that Gab1 in both muscles and terminal Schwann cells is required for proper NMJ development. Gab1 in terminal Schwann cells appeared to regulate the number and process elongation of terminal Schwann cells during synaptic elimination. However, Gab2 knockout mice did not show any defects in the development of the NMJ. Considering the role of Gab1 in postnatal peripheral nerve myelination, our findings suggest that Gab1 is a pleiotropic and important component of NRG1 signals during postnatal development of the peripheral neuromuscular system.

  18. Neuronal Differentiation in Schwann Cell Lineage Underlies Postnatal Neurogenesis in the Enteric Nervous System.

    Science.gov (United States)

    Uesaka, Toshihiro; Nagashimada, Mayumi; Enomoto, Hideki

    2015-07-08

    Elucidation of the cellular identity of neuronal precursors provides mechanistic insights into the development and pathophysiology of the nervous system. In the enteric nervous system (ENS), neurogenesis persists from midgestation to the postnatal period. Cellular mechanism underlying the long-term neurogenesis in the ENS has remained unclear. Using genetic fate mapping in mice, we show here that a subset of Schwann cell precursors (SCPs), which invades the gut alongside the extrinsic nerves, adopts a neuronal fate in the postnatal period and contributes to the ENS. We found SCP-derived neurogenesis in the submucosal region of the small intestine in the absence of vagal neural crest-derived ENS precursors. Under physiological conditions, SCPs comprised up to 20% of enteric neurons in the large intestine and gave rise mainly to restricted neuronal subtypes, calretinin-expressing neurons. Genetic ablation of Ret, the signaling receptor for glial cell line-derived neurotrophic factor, in SCPs caused colonic oligoganglionosis, indicating that SCP-derived neurogenesis is essential to ENS integrity. Identification of Schwann cells as a physiological neurogenic source provides novel insight into the development and disorders of neural crest-derived tissues. Elucidating the cellular identity of neuronal precursors provides novel insights into development and function of the nervous system. The enteric nervous system (ENS) is innervated richly by extrinsic nerve fibers, but little is known about the significance of extrinsic innervation to the structural integrity of the ENS. This report reveals that a subset of Schwann cell precursors (SCPs), which invades the gut alongside the extrinsic nerves, adopts a neuronal fate and differentiates into specific neuronal subtypes. SCP-specific ablation of the Ret gene leads to colonic oligoganglionosis, demonstrating a crucial role of SCP-derived neurogenesis in ENS development. Cross-lineage differentiation capacity in SCPs suggests

  19. Enrichment and Schwann Cell Differentiation of Neural Crest-derived Dental Pulp Stem Cells.

    Science.gov (United States)

    Al-Zer, Heba; Apel, Christian; Heiland, Max; Friedrich, Reinhard E; Jung, Ole; Kroeger, Nadja; Eichhorn, Wolfgang; Smeets, Ralf

    2015-01-01

    As already described in previous studies, neural crest stem cells (NCSCs) can be found in adult human dental pulp. The present study investigated the methodology for enrichment and differentiation-induction of the above mentioned cells. Dental pulp was extracted from human wisdom teeth of four patients and subsequently cultured as explants on fibronectin-coated plates in neurobasal medium supplemented with B27, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin, l-glutamine and neuregulin-β1. The cells were then characterized by immunofluorescence, while their differentiation-potential was tested by the attempt to induce cells into different lineages, i.e. osteogenic, melanocytic and glial. The enriched cell population expressed nestin, CD271 and SOX10, which are well-known markers for NCSCs. Consequently, the cells were successfully induced to differentiate into osteoblasts, melanocytes and Schwann cells, expressing the corresponding differentiation markers. Human adult dental pulp contains a population of stem cells with neural crest ontogeny, which can thus be recruited for multiple regenerative therapies. Copyright © 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  20. Adult skin-derived precursor Schwann cell grafts form growths in the injured spinal cord of Fischer rats.

    Science.gov (United States)

    May, Zacnicte; Kumar, Ranjan; Führmann, Tobias; Tam, Roger; Vulic, Katarina; Forero, Juan; Lucas-Osma, Ana M; Fenrich, Keith; Assinck, Peggy; Lee, Michael J; Moulson, Aaron; Shoichet, Molly S; Tetzlaff, Wolfram; Biernaskie, Jeff; Fouad, Karim

    2017-10-25

    In this study, GFP+ skin-derived precursor Schwann cells (SKP-SCs) from adult rats were grafted into the injured spinal cord of immunosuppressed rats. Our goal was to improve grafted cell survival in the injured spinal cord, which is typically low. Cells were grafted in hyaluronan-methylcellulose hydrogel (HAMC) or hyaluronan-methylcellulose modified with laminin- and fibronectin-derived peptide sequences (eHAMC). The criteria for selection of hyaluronan was for its shear-thinning properties, making the hydrogel easy to inject, methylcellulose for its inverse thermal gelation, helping to keep grafted cells in situ, and fibronectin and laminin to improve cell attachment and, thus, prevent cell death due to dissociation from substrate molecules (i.e., anoikis). Post-mortem examination revealed large masses of GFP+ SKP-SCs in the spinal cords of rats that received cells in HAMC (5 out of n = 8) and eHAMC (6 out of n = 8). Cell transplantation in eHAMC caused significantly greater spinal lesions compared to lesion and eHAMC only control groups. A parallel study showed similar masses in the contused spinal cord of rats after transplantation of adult GFP+ SKP-SCs without a hydrogel or immunosuppression. These findings suggest that adult GFP+ SKP-SCs, cultured/transplanted under the conditions described here, have a capacity for uncontrolled proliferation. Growth-formation in pre-clinical research has also been documented after transplantation of: human induced pluripotent stem cell-derived neural stem cells (Itakura et al., 2015), embryonic stem cells and embryonic stem cell-derived neurons (Brederlau et al., 2006; Dressel et al., 2008), bone marrow derived mesenchymal stem cells (Jeong et al., 2011) and rat nerve-derived SCs following in vitro expansion for ˃11 passages (Funk et al., 2007; Langford et al., 1988; Morrissey et al., 1991). It is of upmost importance to define the precise culture/transplantation parameters for maintenance of normal cell function and safe

  1. Schwann cells promote neuronal differentiation of bone marrow ...

    African Journals Online (AJOL)

    Bone marrow stromal cells (BMSCs), a type of multipotent stem cell, can differentiate into various types of cells. It has been suggested that the BMSCs have the capacity to differentiate into neurons under specific experimental conditions, using chemical factors. In this study, we showed that BMSCs can be induced to ...

  2. Peripheral Nerve Repair with Cultured Schwann Cells: Getting Closer to the Clinics

    Directory of Open Access Journals (Sweden)

    Maria Carolina O. Rodrigues

    2012-01-01

    Full Text Available Peripheral nerve injuries are a frequent and disabling condition, which affects 13 to 23 per 100.000 persons each year. Severe cases, with structural disruption of the nerve, are associated with poor functional recovery. The experimental treatment using nerve grafts to replace damaged or shortened axons is limited by technical difficulties, invasiveness, and mediocre results. Other therapeutic choices include the adjunctive application of cultured Schwann cells and nerve conduits to guide axonal growth. The bone marrow is a rich source of mesenchymal cells, which can be differentiated in vitro into Schwann cells and subsequently engrafted into the damaged nerve. Alternatively, undifferentiated bone marrow mesenchymal cells can be associated with nerve conduits and afterward transplanted. Experimental studies provide evidence of functional, histological, and electromyographical improvement following transplantation of bone-marrow-derived cells in animal models of peripheral nerve injury. This paper focuses on this new therapeutic approach highlighting its direct translational and clinical utility in promoting regeneration of not only acute but perhaps also chronic cases of peripheral nerve damage.

  3. Targeted knock-down of cellular prion protein expression in myelinating Schwann cells does not alter mouse prion pathogenesis.

    Science.gov (United States)

    Halliez, Sophie; Chesnais, Nathalie; Mallucci, Giovanna; Vilotte, Marthe; Langevin, Christelle; Jaumain, Emilie; Laude, Hubert; Vilotte, Jean-Luc; Béringue, Vincent

    2013-06-01

    In naturally acquired transmissible spongiform encephalopathies, the pathogenic agents or prions spread from the sites of initial peripheral uptake or replication to the brain where they cause progressive and fatal neurodegeneration. Routing via the peripheral nervous system is considered to be one of the main pathways to the central nervous system. Replication of prions in Schwann cells is viewed as a potentially important mechanism for efficient prion spread along nerves. Here we used a Cre-loxP mouse transgenetic approach to disrupt host-encoded prion protein (PrP(C)) specifically in myelinating Schwann cells. Despite the use of infection routes targeting highly myelinated nerves, there was no alteration in mouse prion pathogenesis, suggesting that conversion-dependent, centripetal spread of prions does not crucially rely on PrP(C) expressed by myelinating Schwann cells.

  4. Data in support on the shape of Schwann cells and sympathetic neurons onto microconically structured silicon surfaces

    Directory of Open Access Journals (Sweden)

    C. Simitzi

    2015-09-01

    Full Text Available This article contains data related to the research article entitled “Laser fabricated discontinuous anisotropic microconical substrates as a new model scaffold to control the directionality of neuronal network outgrowth” in the Biomaterials journal [1]. Scanning electron microscopy (SEM analysis is performed to investigate whether Schwann cells and sympathetic neurons alter their morphology according to the underlying topography, comprising arrays of silicon microcones with anisotropic geometrical characteristics [1]. It is observed that although soma of sympathetic neurons always preserves its round shape, this is not the case for Schwann cells that become highly polarized in high roughness microconical substrates.

  5. The Proliferation Enhancing Effects of Salidroside on Schwann Cells In Vitro

    Directory of Open Access Journals (Sweden)

    Hui Liu

    2017-01-01

    Full Text Available Derived from Rhodiola rosea L., which is a popular plant in Eastern Europe and Asia, salidroside has pharmacological properties including antiviral, anticancer, hepatoprotective, antidiabetic, and antioxidative effects. Recent studies show that salidroside has neurotrophic and neuroprotective effects. However, the effect of salidroside on Schwann cells (SCs and the underlying mechanisms of the salidroside-induced neurotrophin secretion have seldom been studied. In this study, the effect of salidroside on the survival, proliferation, and gene expression of Schwann cells lineage (RSC96 was studied through the examinations of the cell viability, proliferation, morphology, and expression of neurotrophic factor related genes including BDNF, GDNF, and CDNF at 2, 4, and 6 days, respectively. These results showed that salidroside significantly enhanced survival and proliferation of SCs. The underlying mechanism might involve that salidroside affected SCs growth through the modulation of several neurotrophic factors including BDNF, GDNF, and CDNF. As for the concentration, 0.4 mM, 0.2 mM, and 0.1 mM of salidroside were recommended, especially 0.2 mM. This investigation indicates that salidroside is capable of enhancing SCs survival and function in vitro, which highlights the possibility that salidroside as a drug agent to promote nerve regeneration in cellular nerve scaffold through salidroside-induced neurotrophin secretion in SCs.

  6. The Proliferation Enhancing Effects of Salidroside on Schwann Cells In Vitro.

    Science.gov (United States)

    Liu, Hui; Lv, Peizhen; Wu, Huayu; Zhang, Kun; Xu, Fuben; Zheng, Li; Zhao, Jinmin

    2017-01-01

    Derived from Rhodiola rosea L., which is a popular plant in Eastern Europe and Asia, salidroside has pharmacological properties including antiviral, anticancer, hepatoprotective, antidiabetic, and antioxidative effects. Recent studies show that salidroside has neurotrophic and neuroprotective effects. However, the effect of salidroside on Schwann cells (SCs) and the underlying mechanisms of the salidroside-induced neurotrophin secretion have seldom been studied. In this study, the effect of salidroside on the survival, proliferation, and gene expression of Schwann cells lineage (RSC96) was studied through the examinations of the cell viability, proliferation, morphology, and expression of neurotrophic factor related genes including BDNF, GDNF, and CDNF at 2, 4, and 6 days, respectively. These results showed that salidroside significantly enhanced survival and proliferation of SCs. The underlying mechanism might involve that salidroside affected SCs growth through the modulation of several neurotrophic factors including BDNF, GDNF, and CDNF. As for the concentration, 0.4 mM, 0.2 mM, and 0.1 mM of salidroside were recommended, especially 0.2 mM. This investigation indicates that salidroside is capable of enhancing SCs survival and function in vitro, which highlights the possibility that salidroside as a drug agent to promote nerve regeneration in cellular nerve scaffold through salidroside-induced neurotrophin secretion in SCs.

  7. mTORC1 is essential for early steps during Schwann cell differentiation of amniotic fluid stem cells and regulates lipogenic gene expression.

    Directory of Open Access Journals (Sweden)

    Andrea Preitschopf

    Full Text Available Schwann cell development is hallmarked by the induction of a lipogenic profile. Here we used amniotic fluid stem (AFS cells and focused on the mechanisms occurring during early steps of differentiation along the Schwann cell lineage. Therefore, we initiated Schwann cell differentiation in AFS cells and monitored as well as modulated the activity of the mechanistic target of rapamycin (mTOR pathway, the major regulator of anabolic processes. Our results show that mTOR complex 1 (mTORC1 activity is essential for glial marker expression and expression of Sterol Regulatory Element-Binding Protein (SREBP target genes. Moreover, SREBP target gene activation by statin treatment promoted lipogenic gene expression, induced mTORC1 activation and stimulated Schwann cell differentiation. To investigate mTORC1 downstream signaling we expressed a mutant S6K1, which subsequently induced the expression of the Schwann cell marker S100b, but did not affect lipogenic gene expression. This suggests that S6K1 dependent and independent pathways downstream of mTORC1 drive AFS cells to early Schwann cell differentiation and lipogenic gene expression. In conclusion our results propose that future strategies for peripheral nervous system regeneration will depend on ways to efficiently induce the mTORC1 pathway.

  8. Epi/perineural and Schwann Cells as Well as Perineural Sheath Integrity are Affected Following 2,4-D Exposure.

    Science.gov (United States)

    Sharifi Pasandi, Marzieh; Hosseini Shirazi, Farshad; Gholami, Mohammad Reza; Salehi, Hossein; Najafzadeh, Nowruz; Mazani, Mohammad; Ghasemi Hamidabadi, Hatef; Niapour, Ali

    2017-07-11

    2,4-dicholorophenoxy acetic acid (2,4-D) is a worldwide-known hormone herbicide. However, there are increasing concerns about its exposure and risks of developing pathological conditions for the peripheral nervous system. The aim of this study was to investigate the mechanism(s) involved in the toxicity of 2,4-D on peripheral nerve's cellular components. The epi/perineural and Schwann cells and a total of three cell lines were treated with 2,4-D. The viability of cells at different doses of 2,4-D was measured by MTT assay. The cell cycle analyses, cumulative cell counting, fluorescent staining, antioxidant and caspase enzymes activity were examined on epi/perineural and Schwann cells. The epi/perineural cells were assessed as having biological macromolecular changes. Some tight junction-related genes and proteins were also tested on explants of 2,4-D treated epi/perineural tissue. The viability of 2,4-D treated cells was reduced in a dose-dependent manner. Reduced growth rate and G1 cell cycle arrest were verified in 2,4-D treated epi/perineural and Schwann cells. The use of staining methods (acridine orange/ethidium bromide and DAPI) and caspase 3/7 activity assay along with malondialdehyde, glutathione peroxidase, and superoxide dismutase activity assays indicated the apoptotic and oxidant effects of 2,4-D on epi/perineural and Schwann cells. Data obtained from FTIR revealed changes in epi/perineural proteins and cell membrane lipids. Additionally, claudin-1, occludin, and ZO-1 gene/protein expression profiles were significantly reduced in 2,4-D-treated epi/perineural pieces. Our data indicated that oxidative stress, apoptosis of epi/perineural and Schwann cell and impaired blood-nerve barrier may have contributed to nerve damage following 2,4-D exposure.

  9. Coseeded Schwann cells myelinate neurites from differentiated neural stem cells in neurotrophin-3-loaded PLGA carriers

    Science.gov (United States)

    Xiong, Yi; Zhu, Ji-Xiang; Fang, Zheng-Yu; Zeng, Cheng-Guang; Zhang, Chao; Qi, Guo-Long; Li, Man-Hui; Zhang, Wei; Quan, Da-Ping; Wan, Jun

    2012-01-01

    Biomaterials and neurotrophic factors represent promising guidance for neural repair. In this study, we combined poly-(lactic acid-co-glycolic acid) (PLGA) conduits and neurotrophin-3 (NT-3) to generate NT-3-loaded PLGA carriers in vitro. Bioactive NT-3 was released stably and constantly from PLGA conduits for up to 4 weeks. Neural stem cells (NSCs) and Schwann cells (SCs) were coseeded into an NT-releasing scaffold system and cultured for 14 days. Immunoreactivity against Map2 showed that most of the grafted cells (>80%) were differentiated toward neurons. Double-immunostaining for synaptogenesis and myelination revealed the formation of synaptic structures and myelin sheaths in the coculture, which was also observed under electron microscope. Furthermore, under depolarizing conditions, these synapses were excitable and capable of releasing synaptic vesicles labeled with FM1-43 or FM4-64. Taken together, coseeding NSCs and SCs into NT-3-loaded PLGA carriers increased the differentiation of NSCs into neurons, developed synaptic connections, exhibited synaptic activities, and myelination of neurites by the accompanying SCs. These results provide an experimental basis that supports transplantation of functional neural construction in spinal cord injury. PMID:22619535

  10. Loss of Fig4 in both Schwann cells and motor neurons contributes to CMT4J neuropathy

    Science.gov (United States)

    Vaccari, Ilaria; Carbone, Antonietta; Previtali, Stefano Carlo; Mironova, Yevgeniya A.; Alberizzi, Valeria; Noseda, Roberta; Rivellini, Cristina; Bianchi, Francesca; Del Carro, Ubaldo; D'Antonio, Maurizio; Lenk, Guy M.; Wrabetz, Lawrence; Giger, Roman J.; Meisler, Miriam H.; Bolino, Alessandra

    2015-01-01

    Mutations of FIG4 are responsible for Yunis-Varón syndrome, familial epilepsy with polymicrogyria, and Charcot-Marie-Tooth type 4J neuropathy (CMT4J). Although loss of the FIG4 phospholipid phosphatase consistently causes decreased PtdIns(3,5)P2 levels, cell-specific sensitivity to partial loss of FIG4 function may differentiate FIG4-associated disorders. CMT4J is an autosomal recessive neuropathy characterized by severe demyelination and axonal loss in human, with both motor and sensory involvement. However, it is unclear whether FIG4 has cell autonomous roles in both motor neurons and Schwann cells, and how loss of FIG4/PtdIns(3,5)P2-mediated functions contribute to the pathogenesis of CMT4J. Here, we report that mice with conditional inactivation of Fig4 in motor neurons display neuronal and axonal degeneration. In contrast, conditional inactivation of Fig4 in Schwann cells causes demyelination and defects in autophagy-mediated degradation. Moreover, Fig4-regulated endolysosomal trafficking in Schwann cells is essential for myelin biogenesis during development and for proper regeneration/remyelination after injury. Our data suggest that impaired endolysosomal trafficking in both motor neurons and Schwann cells contributes to CMT4J neuropathy. PMID:25187576

  11. Merlin controls the repair capacity of Schwann cells after injury by regulating Hippo/YAP activity.

    Science.gov (United States)

    Mindos, Thomas; Dun, Xin-Peng; North, Katherine; Doddrell, Robin D S; Schulz, Alexander; Edwards, Philip; Russell, James; Gray, Bethany; Roberts, Sheridan L; Shivane, Aditya; Mortimer, Georgina; Pirie, Melissa; Zhang, Nailing; Pan, Duojia; Morrison, Helen; Parkinson, David B

    2017-02-01

    Loss of the Merlin tumor suppressor and activation of the Hippo signaling pathway play major roles in the control of cell proliferation and tumorigenesis. We have identified completely novel roles for Merlin and the Hippo pathway effector Yes-associated protein (YAP) in the control of Schwann cell (SC) plasticity and peripheral nerve repair after injury. Injury to the peripheral nervous system (PNS) causes a dramatic shift in SC molecular phenotype and the generation of repair-competent SCs, which direct functional repair. We find that loss of Merlin in these cells causes a catastrophic failure of axonal regeneration and remyelination in the PNS. This effect is mediated by activation of YAP expression in Merlin-null SCs, and loss of YAP restores axonal regrowth and functional repair. This work identifies new mechanisms that control the regenerative potential of SCs and gives new insight into understanding the correct control of functional nerve repair in the PNS. © 2017 Mindos et al.

  12. The POU factor Oct-6 is required for the progression of Schwann cell differentiation in peripheral nerves.

    NARCIS (Netherlands)

    M.M. Jaegle (Martine); W.J. Mandemakers (Wim); L.A.M. Broos (Ludo); R. Zwart (Ronald); A. Karis (Alar); P. Visser (Pim); F.G. Grosveld (Frank); D.N. Meijer (Dies)

    1996-01-01

    textabstractThe POU transcription factor Oct-6, also known as SCIP or Tst-1, has been implicated as a major transcriptional regulator in Schwann cell differentiation. Microscopic and immunochemical analysis of sciatic nerves of Oct-6(-/-) mice at different stages of postnatal development reveals a

  13. Schwann Cell Migration Induced by Earthworm Extract via Activation of PAs and MMP2/9 Mediated through ERK1/2 and p38

    Science.gov (United States)

    Chang, Yung-Ming; Shih, Ying-Ting; Chen, Yueh-Sheng; Liu, Chien-Liang; Fang, Wen-Kuei; Tsai, Chang-Hai; Tsai, Fuu-Jen; Kuo, Wei-Wen; Lai, Tung-Yuan; Huang, Chih-Yang

    2011-01-01

    The earthworm, which has stasis removal and wound-healing functions, is a widely used Chinese herbal medicine in China. Schwann cell migration is critical for the regeneration of injured nerves. Schwann cells provide an essentially supportive activity for neuron regeneration. However, the molecular migration mechanisms induced by earthworms in Schwann cells remain unclear. Here, we investigate the roles of MAPK (ERK1/2, JNK and p38) pathways for earthworm-induced matrix-degrading proteolytic enzyme (PAs and MMP2/9) production in Schwann cells. Moreover, earthworm induced phosphorylation of ERK1/2 and p38, but not JNK, activate the downstream signaling expression of PAs and MMPs in a time-dependent manner. Earthworm-stimulated ERK1/2 and p38 phosphorylation was attenuated by pretreatment with U0126 and SB203580, resulting in migration and uPA-related signal pathway inhibition. The results were confirmed using small interfering ERK1/2 and p38 RNA. These results demonstrated that earthworms can stimulate Schwann cell migration and up-regulate PAs and MMP2/9 expression mediated through the MAPK pathways, ERK1/2 and p38. Taken together, our data suggests the MAPKs (ERK1/2, p38)-, PAs (uPA, tPA)-, MMP (MMP2, MMP9) signaling pathway of Schwann cells regulated by earthworms might play a major role in Schwann cell migration and nerve regeneration. PMID:19808845

  14. Neuronal activity in the hub of extrasynaptic Schwann cell-axon interactions.

    Science.gov (United States)

    Samara, Chrysanthi; Poirot, Olivier; Domènech-Estévez, Enric; Chrast, Roman

    2013-01-01

    The integrity and function of neurons depend on their continuous interactions with glial cells. In the peripheral nervous system glial functions are exerted by Schwann cells (SCs). SCs sense synaptic and extrasynaptic manifestations of action potential propagation and adapt their physiology to support neuronal activity. We review here existing literature data on extrasynaptic bidirectional axon-SC communication, focusing particularly on neuronal activity implications. To shed light on underlying mechanisms, we conduct a thorough analysis of microarray data from SC-rich mouse sciatic nerve at different developmental stages and in neuropathic models. We identify molecules that are potentially involved in SC detection of neuronal activity signals inducing subsequent glial responses. We further suggest that alterations in the activity-dependent axon-SC crosstalk impact on peripheral neuropathies. Together with previously reported data, these observations open new perspectives for deciphering glial mechanisms of neuronal function support.

  15. Phenotypic changes of Schwann cells on the proximal stump of injured peripheral nerve during repair using small gap conduit tube

    Directory of Open Access Journals (Sweden)

    Shi-jun Zhang

    2017-01-01

    Full Text Available Dedifferentiation of Schwann cells is an important feature of the response to peripheral nerve injury and specific negative myelination regulators are considered to have a major role in this process. However, most experiments have focused on the distal nerve stump, where the Notch signaling pathway is strongly associated with Schwann cell dedifferentiation and repair of the nerve. We observed the phenotypic changes of Schwann cells and changes of active Notch signaling on the proximal stump during peripheral nerve repair using small gap conduit tubulization. Eighty rats, with right sciatic nerve section of 4 mm, were randomly assigned to conduit bridging group and control group (epineurium suture. Glial fibrillary acidic protein expression, in myelinating Schwann cells on the proximal stump, began to up-regulate at 1 day after injury and was still evident at 5 days. Compared with the control group, Notch1 mRNA was expressed at a higher level in the conduit bridging group during the first week on the proximal stump. Hes1 mRNA levels in the conduit bridging group significantly increased compared with the control group at 3, 5, 7 and 14 days post-surgery. The change of the Notch intracellular domain shared a similar trend as Hes1 mRNA expression. Our results confirmed that phenotypic changes of Schwann cells occurred in the proximal stump. The differences in these changes between the conduit tubulization and epineurium suture groups correlate with changes in Notch signaling. This suggests that active Notch signaling might be a key mechanism during the early stage of neural regeneration in the proximal nerve stump.

  16. Transplantation of neural stem cells, Schwann cells and olfactory ensheathing cells for spinal cord injury : A Web of Science-based literature analysis.

    Science.gov (United States)

    Zhang, Xing; Yin, Fei; Guo, Li; Zhao, Dongxu; Gong, Gu; Gao, Lei; Zhu, Qingsan

    2012-12-15

    To identify global research trends in transplantation of neural stem cells, Schwann cells and olfactory ensheathing cells for spinal cord injury. We performed a bibliometric analysis of studies on transplantation of neural stem cells, Schwann cells and olfactory ensheathing cells for spinal cord injury published from 2002 to 2011 and retrieved from the Web of Science, using the key words spinal cord injury along with either neural stem cell, Schwann cell or olfactory ensheathing cell. (a) peer-reviewed published articles on neural stem cells, Schwann cells or olfactory ensheathing cells for spinal cord injury indexed in the Web of Science; (b) original research articles, reviews, meeting abstracts, proceedings papers, book chapters, editorial materials and news items; and (c) published between 2002 and 2011. (a) articles that required manual searching or telephone access; (b) documents that were not published in the public domain; and (c) corrected papers. (1) Annual publication output, distribution by journal, distribution by institution and top-cited articles on neural stem cells; (2) annual publication output, distribution by journal, distribution by institution and top-cited articles on Schwann cells; (3) annual publication output, distribution by journal, distribution by institution and top-cited articles on olfactory ensheathing cells. This analysis, based on articles indexed in the Web of Science, identified several research trends among studies published over the past 10 years in transplantation of neural stem cells, Schwann cells and olfactory ensheathing cells for spinal cord injury. The number of publications increased over the 10-year period examined. Most papers appeared in journals with a focus on neurology, such as Journal of Neurotrauma, Experimental Neurology and Glia. Research institutes publishing on the use of neural stem cells to repair spinal cord injury were mostly in the USA and Canada. Those publishing on the use of Schwann cells were

  17. Tead1 regulates the expression of Peripheral Myelin Protein 22 during Schwann cell development.

    Science.gov (United States)

    Lopez-Anido, Camila; Poitelon, Yannick; Gopinath, Chetna; Moran, John J; Ma, Ki Hwan; Law, William D; Antonellis, Anthony; Feltri, M Laura; Svaren, John

    2016-07-15

    Schwann cells are myelinating glia in the peripheral nervous system that form the myelin sheath. A major cause of peripheral neuropathy is a copy number variant involving the Peripheral Myelin Protein 22 (PMP22) gene, which is located within a 1.4-Mb duplication on chromosome 17 associated with the most common form of Charcot-Marie-Tooth Disease (CMT1A). Rodent models of CMT1A have been used to show that reducing Pmp22 overexpression mitigates several aspects of a CMT1A-related phenotype. Mechanistic studies of Pmp22 regulation identified enhancers regulated by the Sox10 (SRY sex determining region Y-box 10) and Egr2/Krox20 (Early growth response protein 2) transcription factors in myelinated nerves. However, relatively little is known regarding how other transcription factors induce Pmp22 expression during Schwann cell development and myelination. Here, we examined Pmp22 enhancers as a function of cell type-specificity, nerve injury and development. While Pmp22 enhancers marked by active histone modifications were lost or remodeled after injury, we found that these enhancers were permissive in early development prior to Pmp22 upregulation. Pmp22 enhancers contain binding motifs for TEA domain (Tead) transcription factors of the Hippo signaling pathway. We discovered that Tead1 and co-activators Yap and Taz are required for Pmp22 expression, as well as for the expression of Egr2 Tead1 directly binds Pmp22 and Egr2 enhancers early in development and Tead1 binding is induced during myelination, correlating with Pmp22 expression. The data identify Tead1 as a novel regulator of Pmp22 expression during development in concert with Sox10 and Egr2. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  18. The insulin-like growth factors I and II stimulate proliferation of different types of Schwann cells

    DEFF Research Database (Denmark)

    Sondell, M; Svenningsen, Åsa Fex; Kanje, M

    1997-01-01

    A combination of immunocytochemistry for glial specific antigens and bromodeoxyuridine (BrdU) and teasing was used to identify proliferating cells in cultured rat sciatic nerve segments. The nerve segments were exposed to insulin, or the insulin-like growth factors IGF-I and IGF-II. Teasing in co......, truncated IGF-I promoted proliferation of Schwann cells of myelinated nerve fibres while insulin increased proliferation of both cell types....

  19. Embryonic Stem Cells: Isolation, Characterization and Culture

    Science.gov (United States)

    Amit, Michal; Itskovitz-Eldor, Joseph

    Embryonic stem cells are pluripotent cells isolated from the mammalian blastocyst. Traditionally, these cells have been derived and cultured with mouse embryonic fibroblast (MEF) supportive layers, which allow their continuous growth in an undifferentiated state. However, for any future industrial or clinical application hESCs should be cultured in reproducible, defined, and xeno-free culture system, where exposure to animal pathogens is prevented. From their derivation in 1998 the methods for culturing hESCs were significantly improved. This chapter wills discuss hESC characterization and the basic methods for their derivation and maintenance.

  20. Transcriptome Landscapes of Mammalian Embryonic Cells

    NARCIS (Netherlands)

    Brinkhof, B.

    2015-01-01

    This thesis describes research on gene expression profiles from different embryonic stages and cell types to identify genes involved in pluripotency or differentiation in bovine and porcine cells. The results are compared with data from other mammals. RNA expression profiles of morula and blastocyst

  1. Essential and distinct roles for cdc42 and rac1 in the regulation of Schwann cell biology during peripheral nervous system development

    DEFF Research Database (Denmark)

    Benninger, Yves; Thurnherr, Tina; Pereira, Jorge A

    2007-01-01

    During peripheral nervous system (PNS) myelination, Schwann cells must interpret extracellular cues to sense their environment and regulate their intrinsic developmental program accordingly. The pathways and mechanisms involved in this process are only partially understood. We use tissue-specific...

  2. Embryonic stem cells: testing the germ-cell theory.

    Science.gov (United States)

    Hochedlinger, Konrad

    2011-10-25

    The exact cellular origin of embryonic stem cells remains elusive. Now a new study provides compelling evidence that embryonic stem cells, established under conventional culture conditions, originate from a transient germ-cell state. Copyright © 2011 Elsevier Ltd. All rights reserved.

  3. Schwann Cell and Axon: An Interlaced Unit-From Action Potential to Phenotype Expression.

    Science.gov (United States)

    Court, Felipe A; Alvarez, Jaime

    2016-01-01

    Here we propose a model of a peripheral axon with a great deal of autonomy from its cell body-the autonomous axon-but with a substantial dependence on its ensheathing Schwann cell (SC), the axon-SC unit. We review evidence in several fields and show that (i) axons can extend sprouts and grow without the concurrence of the cell body, but regulated by SCs; (ii) axons synthesize their proteins assisted by SCs that supply them with ribosomes and, probably, with mRNAs by way of exosomes; (iii) the molecular organization of the axoplasm, i.e., its phenotype, is regulated by the SC, as illustrated by the axonal microtubular content, which is down-regulated by the SC; and (iv) the axon has a program for self-destruction that is boosted by the SC. The main novelty of this model axon-SC unit is that it breaks with the notion that all proteins of the nerve cell are specified by its own nucleus. The notion of a collaborative specification of the axoplasm by more than one nucleus, which we present here, opens a new dimension in the understanding of the nervous system in health and disease and is also a frame of reference to understand other tissues or cell associations.

  4. Human autologous serum as a substitute for fetal bovine serum in human Schwann cell culture.

    Directory of Open Access Journals (Sweden)

    Parisa Goodarzi

    2014-04-01

    Full Text Available Nowadays, cell -based and tissue engineered products have opened new horizons in treatment of incurable nervous system disorders. The number of studies on the role of Schwann cells (SC in treating nervous disorders is higher than other cell types. Different protocols have been suggested for isolation and expansion of SC which most of them have used multiple growth factors, mitogens and fetal bovine sera (FBS in culture medium. Because of potential hazards of animal-derived reagents, this study was designed to evaluate the effect of replacing FBS with human autologous serum (HAS on SC's yield and culture parameters. Samples from 10 peripheral nerve biopsies were retrieved and processed under aseptic condition. The isolated cells cultured in FBS (1st group or autologous serum (2nd group. After primary culture the cells were seeded at 10000 cell/cm2 in a 12 wells cell culture plate for each group. At 100% confluency, the cell culture parameters (count, viability, purity and culture duration of 2 groups were compared using paired t-test. The average donors' age was 35.80 (SD=13.35 and except for 1 sample the others cultured successfully. In first group, the averages of cell purity, viability and culture duration were 97% (SD=1.32, 97/33% (SD=1.22 and 11.77 (SD=2.58 days respectively. This parameters were 97.33% (SD=1.00, 97.55% (SD=1.33 and 10.33 days (SD=1.65 in second group. The difference of cell count, purity and viability were not significant between 2 groups (P>0.05. The cells of second group reached to 100% confluency in shorter period of time (P=0.03. The results of this study showed that autologous serum can be a good substitute for FBS in human SC culture. This can reduce the costs and improve the safety of cell product for clinical application.

  5. Mycobacterium leprae downregulates the expression of PHEX in Schwann cells and osteoblasts

    Directory of Open Access Journals (Sweden)

    Sandra R Boiça Silva

    2010-08-01

    Full Text Available Neuropathy and bone deformities, lifelong sequelae of leprosy that persist after treatment, result in significant impairment to patients and compromise their social rehabilitation. Phosphate-regulating gene with homologies to endopeptidase on the X chromosome (PHEX is a Zn-metalloendopeptidase, which is abundantly expressed in osteoblasts and many other cell types, such as Schwann cells, and has been implicated in phosphate metabolism and X-linked rickets. Here, we demonstrate that Mycobacterium leprae stimulation downregulates PHEX transcription and protein expression in a human schwannoma cell line (ST88-14 and human osteoblast lineage. Modulation of PHEX expression was observed to a lesser extent in cells stimulated with other species of mycobacteria, but was not observed in cultures treated with latex beads or with the facultative intracellular bacterium Salmonella typhimurium. Direct downregulation of PHEX by M. leprae could be involved in the bone resorption observed in leprosy patients. This is the first report to describe PHEX modulation by an infectious agent.

  6. Preparation of graphene oxide/polyacrylamide composite hydrogel and its effect on Schwann cells attachment and proliferation.

    Science.gov (United States)

    Li, Guicai; Zhao, Yinxin; Zhang, Luzhong; Gao, Ming; Kong, Yan; Yang, Yumin

    2016-07-01

    Various hydrogel materials have been developed for improving the regeneration of peripheral nerve. Among which the graphene related hydrogels with excellent mechanical properties have attracted great attention. However, the effect of these hydrogels on peripheral nerve regeneration is still unclear. In the present study, the graphene oxide/polyacrylamide (GO/PAM) composite hydrogels were fabricated by in-situ free radical polymerization. The morphology, wettability, composition, swelling ratio, mechanical property and degradation behavior of the prepared GO/PAM composite hydrogels were separately characterized. The effect of GO/PAM hydrogel on the attachment and proliferation of Schwann cells was evaluated. Moreover, the release of biofactors by Schwann cells and adsorption of matrix proteins were further measured. The results showed that the color of the hydrogel became darker with the increased GO concentration, while the surface pore structure also displayed large variation when GO concentration was increased. The hydrophobicity and mechanical properties of hydrogel were increased with the ascending GO concentration. In addition, the variation of GO concentration displayed no obvious influence on the degradation of the composite hydrogel in different medium. The GO/PAM composite hydrogel with 0.4% GO (G0.4) could effectively enhance the attachment and proliferation of Schwann cells. Furthermore, the cells on G0.4 hydrogel displayed higher biofactors release and larger matrix adsorption than other samples. The results demonstrated that GO with suitable concentration in PAM hydrogel could effectively promote Schwann cell growth. The study may provide an important experimental basis for the design and development of new nerve grafts with potential application for peripheral nerve regeneration. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Autophagy in human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Thien Tra

    Full Text Available Autophagy (macroautophagy is a degradative process that involves the sequestration of cytosolic material including organelles into double membrane vesicles termed autophagosomes for delivery to the lysosome. Autophagy is essential for preimplantation development of mouse embryos and cavitation of embryoid bodies. The precise roles of autophagy during early human embryonic development, remain however largely uncharacterized. Since human embryonic stem cells constitute a unique model system to study early human embryogenesis we investigated the occurrence of autophagy in human embryonic stem cells. We have, using lentiviral transduction, established multiple human embryonic stem cell lines that stably express GFP-LC3, a fluorescent marker for the autophagosome. Each cell line displays both a normal karyotype and pluripotency as indicated by the presence of cell types representative of the three germlayers in derived teratomas. GFP expression and labelling of autophagosomes is retained after differentiation. Baseline levels of autophagy detected in cultured undifferentiated hESC were increased or decreased in the presence of rapamycin and wortmannin, respectively. Interestingly, autophagy was upregulated in hESCs induced to undergo differentiation by treatment with type I TGF-beta receptor inhibitor SB431542 or removal of MEF secreted maintenance factors. In conclusion we have established hESCs capable of reporting macroautophagy and identify a novel link between autophagy and early differentiation events in hESC.

  8. Schwann cells are activated by ATP released from neurons in an in vitro cellular model of Miller Fisher syndrome.

    Science.gov (United States)

    Rodella, Umberto; Negro, Samuele; Scorzeto, Michele; Bergamin, Elisanna; Jalink, Kees; Montecucco, Cesare; Yuki, Nobuhiro; Rigoni, Michela

    2017-05-01

    The neuromuscular junction is exposed to different types of insult, including mechanical trauma, toxins and autoimmune antibodies and, accordingly, has retained through evolution a remarkable ability to regenerate. Regeneration is driven by multiple signals that are exchanged among the cellular components of the junction. These signals are largely unknown. Miller Fisher syndrome is a variant of Guillain-Barré syndrome caused by autoimmune antibodies specific for epitopes of peripheral axon terminals. Using an animal model of Miller Fisher syndrome, we recently reported that a monoclonal anti-polysialoganglioside GQ1b antibody plus complement damages nerve terminals with production of mitochondrial hydrogen peroxide, which activates Schwann cells. Several additional signaling molecules are likely to be involved in the activation of the regeneration program in these cells. Using an in vitro cellular model consisting of co-cultured primary neurons and Schwann cells, we found that ATP is released by neurons injured by the anti-GQ1b antibody plus complement. Neuron-derived ATP acts as an alarm messenger for Schwann cells, where it induces the activation of intracellular pathways, including calcium signaling, cAMP and CREB, which, in turn, produce signals that promote nerve regeneration. These results contribute to defining the cross-talk taking place at the neuromuscular junction when it is attacked by anti-gangliosides autoantibodies plus complement, which is crucial for nerve regeneration and is also likely to be important in other peripheral neuropathies. © 2017. Published by The Company of Biologists Ltd.

  9. Schwann cells are activated by ATP released from neurons in an in vitro cellular model of Miller Fisher syndrome

    Directory of Open Access Journals (Sweden)

    Umberto Rodella

    2017-05-01

    Full Text Available The neuromuscular junction is exposed to different types of insult, including mechanical trauma, toxins and autoimmune antibodies and, accordingly, has retained through evolution a remarkable ability to regenerate. Regeneration is driven by multiple signals that are exchanged among the cellular components of the junction. These signals are largely unknown. Miller Fisher syndrome is a variant of Guillain–Barré syndrome caused by autoimmune antibodies specific for epitopes of peripheral axon terminals. Using an animal model of Miller Fisher syndrome, we recently reported that a monoclonal anti-polysialoganglioside GQ1b antibody plus complement damages nerve terminals with production of mitochondrial hydrogen peroxide, which activates Schwann cells. Several additional signaling molecules are likely to be involved in the activation of the regeneration program in these cells. Using an in vitro cellular model consisting of co-cultured primary neurons and Schwann cells, we found that ATP is released by neurons injured by the anti-GQ1b antibody plus complement. Neuron-derived ATP acts as an alarm messenger for Schwann cells, where it induces the activation of intracellular pathways, including calcium signaling, cAMP and CREB, which, in turn, produce signals that promote nerve regeneration. These results contribute to defining the cross-talk taking place at the neuromuscular junction when it is attacked by anti-gangliosides autoantibodies plus complement, which is crucial for nerve regeneration and is also likely to be important in other peripheral neuropathies.

  10. Ponatinib promotes a G1 cell-cycle arrest of merlin/NF2-deficient human schwann cells.

    Science.gov (United States)

    Petrilli, Alejandra M; Garcia, Jeanine; Bott, Marga; Klingeman Plati, Stephani; Dinh, Christine T; Bracho, Olena R; Yan, Denise; Zou, Bing; Mittal, Rahul; Telischi, Fred F; Liu, Xue-Zhong; Chang, Long-Sheng; Welling, D Bradley; Copik, Alicja J; Fernández-Valle, Cristina

    2017-05-09

    Neurofibromatosis type 2 (NF2) is a genetic syndrome that predisposes individuals to multiple benign tumors of the central and peripheral nervous systems, including vestibular schwannomas. Currently, there are no FDA approved drug therapies for NF2. Loss of function of merlin encoded by the NF2 tumor suppressor gene leads to activation of multiple mitogenic signaling cascades, including platelet-derived growth factor receptor (PDGFR) and SRC in Schwann cells. The goal of this study was to determine whether ponatinib, an FDA-approved ABL/SRC inhibitor, reduced proliferation and/or survival of merlin-deficient human Schwann cells (HSC). Merlin-deficient HSC had higher levels of phosphorylated PDGFRα/β, and SRC than merlin-expressing HSC. A similar phosphorylation pattern was observed in phospho-protein arrays of human vestibular schwannoma samples compared to normal HSC. Ponatinib reduced merlin-deficient HSC viability in a dose-dependent manner by decreasing phosphorylation of PDGFRα/β, AKT, p70S6K, MEK1/2, ERK1/2 and STAT3. These changes were associated with decreased cyclin D1 and increased p27Kip1levels, leading to a G1 cell-cycle arrest as assessed by Western blotting and flow cytometry. Ponatinib did not modulate ABL, SRC, focal adhesion kinase (FAK), or paxillin phosphorylation levels. These results suggest that ponatinib is a potential therapeutic agent for NF2-associated schwannomas and warrants further in vivo investigation.

  11. Embryonic Stem Cells and their Genetic Modification

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 13; Issue 2. Embryonic Stem Cells and their Genetic Modification - The Nobel Prize in Physiology or Medicine 2007. Mitradas M Panicker. General Article Volume 13 Issue 2 February 2008 pp 172-180 ...

  12. Cytokine signalling in embryonic stem cells

    DEFF Research Database (Denmark)

    Kristensen, David Møbjerg; Kalisz, Mark; Nielsen, Jens Høiriis

    2006-01-01

    Cytokines play a central role in maintaining self-renewal in mouse embryonic stem (ES) cells through a member of the interleukin-6 type cytokine family termed leukemia inhibitory factor (LIF). LIF activates the JAK-STAT3 pathway through the class I cytokine receptor gp130, which forms a trimeric...... pathways seem to converge on c-myc as a common target to promote self-renewal. Whereas LIF does not seem to stimulate self-renewal in human embryonic stem cells it cannot be excluded that other cytokines are involved. The pleiotropic actions of the increasing number of cytokines and receptors signalling...... via JAKs, STATs and SOCS exhibit considerable redundancy, compensation and plasticity in stem cells in accordance with the view that stem cells are governed by quantitative variations in strength and duration of signalling events known from other cell types rather than qualitatively different stem...

  13. Exogenous schwann cells migrate, remyelinate and promote clinical recovery in experimental auto-immune encephalomyelitis.

    Directory of Open Access Journals (Sweden)

    Violetta Zujovic

    Full Text Available Schwann cell (SC transplantation is currently being discussed as a strategy that may promote functional recovery in patients with multiple sclerosis (MS and other inflammatory demyelinating diseases of the central nervous system (CNS. However this assumes they will not only survive but also remyelinate demyelinated axons in the chronically inflamed CNS. To address this question we investigated the fate of transplanted SCs in myelin oligodendrocyte glycoprotein (MOG-induced experimental autoimmune encephalomyelitis (EAE in the Dark Agouti rat; an animal model that reproduces the complex inflammatory demyelinating immunopathology of MS. We now report that SCs expressing green fluorescent protein (GFP-SCs allografted after disease onset not only survive but also migrate to remyelinate lesions in the inflamed CNS. GFP-SCs were detected more frequently in the parenchyma after direct injection into the spinal cord, than via intra-thecal delivery into the cerebrospinal fluid. In both cases the transplanted cells intermingled with astrocytes in demyelinated lesions, aligned with axons and by twenty one days post transplantation had formed Pzero protein immunoreactive internodes. Strikingly, GFP-SCs transplantation was associated with marked decrease in clinical disease severity in terms of mortality; all GFP-SCs transplanted animals survived whilst 80% of controls died within 40 days of disease.

  14. Exogenous Schwann Cells Migrate, Remyelinate and Promote Clinical Recovery in Experimental Auto-Immune Encephalomyelitis

    Science.gov (United States)

    Zujovic, Violetta; Doucerain, Cédric; Hidalgo, Antoine; Bachelin, Corinne; Lachapelle, François; Weissert, Robert; Stadelmann, Christine; Linington, Chris; Evercooren, Anne Baron-Van

    2012-01-01

    Schwann cell (SC) transplantation is currently being discussed as a strategy that may promote functional recovery in patients with multiple sclerosis (MS) and other inflammatory demyelinating diseases of the central nervous system (CNS). However this assumes they will not only survive but also remyelinate demyelinated axons in the chronically inflamed CNS. To address this question we investigated the fate of transplanted SCs in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) in the Dark Agouti rat; an animal model that reproduces the complex inflammatory demyelinating immunopathology of MS. We now report that SCs expressing green fluorescent protein (GFP-SCs) allografted after disease onset not only survive but also migrate to remyelinate lesions in the inflamed CNS. GFP-SCs were detected more frequently in the parenchyma after direct injection into the spinal cord, than via intra-thecal delivery into the cerebrospinal fluid. In both cases the transplanted cells intermingled with astrocytes in demyelinated lesions, aligned with axons and by twenty one days post transplantation had formed Pzero protein immunoreactive internodes. Strikingly, GFP-SCs transplantation was associated with marked decrease in clinical disease severity in terms of mortality; all GFP-SCs transplanted animals survived whilst 80% of controls died within 40 days of disease. PMID:22984406

  15. Inhibition of SIRT2 in merlin/NF2-mutant Schwann cells triggers necrosis.

    Science.gov (United States)

    Petrilli, Alejandra; Bott, Marga; Fernández-Valle, Cristina

    2013-12-01

    Mutations in the NF2 gene cause Neurofibromatosis Type 2 (NF2), a disorder characterized by the development of schwannomas, meningiomas and ependymomas in the nervous system. Merlin, a tumor suppressor encoded by the NF2 gene, modulates activity of many essential signaling pathways. Yet despite increasing knowledge of merlin function, there are no NF2 drug therapies. In a pilot high-throughput screen of the Library of Pharmacologically Active Compounds, we assayed for compounds capable of reducing viability of mouse Schwann cells (MSC) with Nf2 inactivation as a cellular model for human NF2 schwannomas. AGK2, a SIRT2 (sirtuin 2) inhibitor, was identified as a candidate compound. SIRT2 is one of seven mammalian sirtuins that are NAD+-dependent protein deacetylases. We show that merlin-mutant MSC have higher expression levels of SIRT2 and lower levels of overall lysine acetylation than wild-type control MSC. Pharmacological inhibition of SIRT2 decreases merlin-mutant MSC viability in a dose dependent manner without substantially reducing wild-type MSC viability. Inhibition of SIRT2 activity in merlin-mutant MSC is accompanied by release of lactate dehydrogenase and high mobility group box 1 protein into the medium in the absence of significant apoptosis, autophagy, or cell cycle arrest. These findings suggest that SIRT2 inhibition triggers necrosis of merlin-mutant MSCs and that SIRT2 is a potential NF2 drug target.

  16. Schwann cell transplantation and descending propriospinal regeneration after spinal cord injury.

    Science.gov (United States)

    Deng, Ling-Xiao; Walker, Chandler; Xu, Xiao-Ming

    2015-09-04

    After spinal cord injury (SCI), poor ability of damaged axons of the central nervous system (CNS) to regenerate causes very limited functional recovery. Schwann cells (SCs) have been widely explored as promising donors for transplantation to promote axonal regeneration in the CNS including the spinal cord. Compared with other CNS axonal pathways, injured propriospinal tracts display the strongest regenerative response to SC transplantation. Even without providing additional neurotrophic factors, propriospinal axons can grow into the SC environment which is rarely seen in supraspinal tracts. Propriospinal tract has been found to respond to several important neurotrophic factors secreted by SCs. Therefore, the SC is considered to be one of the most promising candidates for cell-based therapies for SCI. Since many reviews have already appeared on topics of SC transplantation in SCI repair, this review will focus particularly on the rationale of SC transplantation in mediating descending propriospinal axonal regeneration as well as optimizing such regeneration by using different combinatorial strategies. This article is part of a Special Issue entitled SI: Spinal cord injury. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. A new electrospun graphene-silk fibroin composite scaffolds for guiding Schwann cells.

    Science.gov (United States)

    Zhao, Yahong; Gong, Jiahuan; Niu, Changmei; Wei, Ziwei; Shi, Jiaqi; Li, Guohui; Yang, Yumin; Wang, Hongbo

    2017-12-01

    Graphene (Gr) has been made of various forms used for repairing peripheral nerve injury with favorable electroactivity, however, graphene-based scaffolds in peripheral nerve regeneration are still rarely reported due to the difficulty of realizing uniform dispersion of graphene and electroactive materials at nanoscale as well as lacking biocompatibility. In this paper, graphene-silk fibroin (SF) composite nanofiber membranes with different mass ratios were prepared via electrospinning. Microscopic observation revealed that electrospun Gr/SF membranes had a nanofibrous structure. Electrochemical analysis provided electroactivity characterization of the Gr/SF membranes. The physiochemical results showed that the physiochemical properties of electrospun Gr/SF membranes could be changed by varying Gr concentration. Swelling ratio and contact angle measurements confirmed that electrospun Gr/SF membranes possessed large absorption capacity and hydrophilic surface, and the mechanical property was improved with increasing Gr concentration. Additionally, in-vitro cytotoxicity with L929 revealed that all the electrospun Gr/SF membranes are biocompatible. Moreover, the morphology and quantity showed that the membranes supported the survival and growth of the cultured Schwann cells. Collectively, all of the results suggest that the electrospun Gr/SF membranes combine the excellent electrically conductivity and mechanical strength of the graphene with biocompatibility property of silk to mimic the natural neural cell micro-environment for nerve development.

  18. Hierarchical thermoplastic rippled nanostructures regulate Schwann cell adhesion, morphology and spatial organization.

    Science.gov (United States)

    Masciullo, Cecilia; Dell'Anna, Rossana; Tonazzini, Ilaria; Böettger, Roman; Pepponi, Giancarlo; Cecchini, Marco

    2017-10-12

    Periodic ripples are a variety of anisotropic nanostructures that can be realized by ion beam irradiation on a wide range of solid surfaces. Only a few authors have investigated these surfaces for tuning the response of biological systems, probably because it is challenging to directly produce them in materials that well sustain long-term cellular cultures. Here, hierarchical rippled nanotopographies with a lateral periodicity of ∼300 nm are produced from a gold-irradiated germanium mold in polyethylene terephthalate (PET), a biocompatible polymer approved by the US Food and Drug Administration for clinical applications, by a novel three-step embossing process. The effects of nano-ripples on Schwann Cells (SCs) are studied in view of their possible use for nerve-repair applications. The data demonstrate that nano-ripples can enhance short-term SC adhesion and proliferation (3-24 h after seeding), drive their actin cytoskeleton spatial organization and sustain long-term cell growth. Notably, SCs are oriented perpendicularly with respect to the nanopattern lines. These results provide information about the possible use of hierarchical nano-rippled elements for nerve-regeneration protocols.

  19. Puerarin may protect against Schwann cell damage induced by glucose fluctuation.

    Science.gov (United States)

    Xue, Bing; Wang, Lin; Zhang, Zhe; Wang, Rui; Xia, Xin-Xin; Han, Ping-Ping; Cao, Li-Jun; Liu, Yong-Hui; Sun, Lian-Qing

    2017-07-01

    Puerarin is one of the major active ingredients in Gegen, a traditional Chinese herb that has been reported to have a wide variety of beneficial pharmacology functions. Previous studies have implicated that the damaging effects of hyperglycemia resulting from oxidative stress and glucose fluctuation may be more dangerous than constant high glucose in the development of diabetes-related complications. The present study focuses on the effects of puerarin on glucose fluctuation-induced oxidative stress-induced Schwann cell (SC) apoptosis in vitro. Primarily cultured SCs were exposed to different conditions and the effect of puerarin on cell viability was determined by MTT assays. Intracellular reactive oxygen species (ROS) generation and mitochondrial transmembrane potential were detected by flow cytometry analysis. Apoptosis was confirmed by the Annexin V-FITC/PI and TUNEL method. Quantitative real-time reverse transcriptase polymerase chain reaction was performed to analyze the expression levels of bax and bcl-2. Western blot was performed to analyze the expression levels of some important transcription factors and proteins. The results showed that incubating SCs with intermittent high glucose for 48 h decreased cell viability and increased the number of apoptotic cells whereas treating with puerarin protected SCs against glucose fluctuation-induced cell damage. Further study demonstrated that puerarin suppressed activation of apoptosis-related proteins including PARP and caspase-3, downregulation of bcl-2, and upregulation of intracellular distribution of bax from cytosol to mitochondria, which was induced by glucose fluctuation. Moreover, puerarin inhibited the elevation of intracellular ROS and mitochondrial depolarization induced by glucose fluctuation. These results suggest that puerarin may protect SCs against glucose fluctuation-induced cell injury through inhibiting apoptosis as well as oxidative stress.

  20. Lentiviral-mediated transfer of CNTF to schwann cells within reconstructed peripheral nerve grafts enhances adult retinal ganglion cell survival and axonal regeneration

    NARCIS (Netherlands)

    Hu, Ying; Leaver, Simone G; Plant, Giles W; Hendriks, William T J; Niclou, Simone P; Verhaagen, J.; Harvey, Alan R; Cui, Qi

    We recently described a method for reconstituting peripheral nerve (PN) sheaths using adult Schwann cells (SCs). Reconstructed PN tissue grafted onto the cut optic nerve supports the regeneration of injured adult rat retinal ganglion cell (RGC) axons. To determine whether genetic manipulation of

  1. Collagen nerve conduits promote enhanced axonal regeneration, schwann cell association, and neovascularization compared to silicone conduits.

    Science.gov (United States)

    Kemp, Stephen W P; Syed, Shahbaz; Walsh, Walsh; Zochodne, Douglas W; Midha, Rajiv

    2009-08-01

    Peripheral nerve regeneration within guidance conduits involves a critical association between regenerating axons, Schwann cells (SCs), and neovascularization. However, it is currently unknown if there is a greater association between these factors in nonpermeable versus semipermeable nerve guide conduits. We therefore examined this collaboration in both silicone- and collagen-based nerve conduits in both 5- and 10-mm-injury gaps in rat sciatic nerves. Results indicate that collagen conduits promoted enhanced axonal and SC regeneration and association when compared to silicone conduits in the shorter 5-mm-gap model. In addition, collagen tubes displayed enhanced neovascularization over silicone conduits, suggesting that these three factors are intimately related in successful peripheral nerve regeneration. At later time points (1- and 2-month analysis) in a 10-mm-gap model, collagen tubes displayed enhanced axonal regeneration, myelination, and vascularization when compared to silicone-based conduits. Results from these studies suggest that regenerating cables within collagen-based conduits are revascularized earlier and more completely, which in turn enhances peripheral nerve regeneration through these nerve guides as compared to silicone conduits.

  2. Schwann cell mitochondria as key regulators in the development and maintenance of peripheral nerve axons.

    Science.gov (United States)

    Ino, Daisuke; Iino, Masamitsu

    2017-03-01

    Formation of myelin sheaths by Schwann cells (SCs) enables rapid and efficient transmission of action potentials in peripheral axons, and disruption of myelination results in disorders that involve decreased sensory and motor functions. Given that construction of SC myelin requires high levels of lipid and protein synthesis, mitochondria, which are pivotal in cellular metabolism, may be potential regulators of the formation and maintenance of SC myelin. Supporting this notion, abnormal mitochondria are found in SCs of neuropathic peripheral nerves in both human patients and the relevant animal models. However, evidence for the importance of SC mitochondria in myelination has been limited, until recently. Several studies have recently used genetic approaches that allow SC-specific ablation of mitochondrial metabolic activity in living animals to show the critical roles of SC mitochondria in the development and maintenance of peripheral nerve axons. Here, we review current knowledge about the involvement of SC mitochondria in the formation and dysfunction of myelinated axons in the peripheral nervous system.

  3. Permissive Schwann cell graft/spinal cord interfaces for axon regeneration.

    Science.gov (United States)

    Williams, Ryan R; Henao, Martha; Pearse, Damien D; Bunge, Mary Bartlett

    2015-01-01

    The transplantation of autologous Schwann cells (SCs) to repair the injured spinal cord is currently being evaluated in a clinical trial. In support, this study determined properties of spinal cord/SC bridge interfaces that enabled regenerated brainstem axons to cross them, possibly leading to improvement in rat hindlimb movement. Fluid bridges of SCs and Matrigel were placed in complete spinal cord transections. Compared to pregelled bridges of SCs and Matrigel, they improved regeneration of brainstem axons across the rostral interface. The regenerating brainstem axons formed synaptophysin(+) bouton-like terminals and contacted MAP2A(+) dendrites at the caudal interface. Brainstem axon regeneration was directly associated with glial fibrillary acidic protein (GFAP(+)) astrocyte processes that elongated into the SC bridge. Electron microscopy revealed that axons, SCs, and astrocytes were enclosed together within tunnels bounded by a continuous basal lamina. Neuroglycan (NG2) expression was associated with these tunnels. One week after injury, the GFAP(+) processes coexpressed nestin and brain lipid-binding protein, and the tips of GFAP(+)/NG2(+) processes extended into the bridges together with the regenerating brainstem axons. Both brainstem axon regeneration and number of GFAP(+) processes in the bridges correlated with improvement in hindlimb locomotion. Following SCI, astrocytes may enter a reactive state that prohibits axon regeneration. Elongation of astrocyte processes into SC bridges, however, and formation of NG2(+) tunnels enable brainstem axon regeneration and improvement in function. It is important for spinal cord repair to define conditions that favor elongation of astrocytes into lesions/transplants.

  4. Implications of Schwann Cells Biomechanics and Mechanosensitivity for Peripheral Nervous System Physiology and Pathophysiology

    Directory of Open Access Journals (Sweden)

    Gonzalo Rosso

    2017-10-01

    Full Text Available The presence of bones around the central nervous system (CNS provides it with highly effective physiologically crucial mechanical protection. The peripheral nervous system (PNS, in contrast, lacks this barrier. Consequently, the long held belief is that the PNS is mechanically vulnerable. On the other hand, the PNS is exposed to a variety of physiological mechanical stresses during regular daily activities. This fact prompts us to question the dogma of PNS mechanical vulnerability. As a matter of fact, impaired mechanics of PNS nerves is associated with neuropathies with the liability to mechanical stresses paralleled by significant impairment of PNS physiological functions. Our recent biomechanical integrity investigations on nerve fibers from wild-type and neuropathic mice lend strong support in favor of natural mechanical protection of the PNS and demonstrate a key role of Schwann cells (SCs therein. Moreover, recent works point out that SCs can sense mechanical properties of their microenvironment and the evidence is growing that SCs mechanosensitivity is important for PNS development and myelination. Hence, SCs exhibit mechanical strength necessary for PNS mechanoprotection as well as mechanosensitivity necessary for PNS development and myelination. This mini review reflects on the intriguing dual ability of SCs and implications for PNS physiology and pathophysiology.

  5. Epalrestat increases intracellular glutathione levels in Schwann cells through transcription regulation

    Directory of Open Access Journals (Sweden)

    Keisuke Sato

    2014-01-01

    Full Text Available Epalrestat (EPS, approved in Japan, is the only aldose reductase inhibitor that is currently available for the treatment of diabetic neuropathy. Here we report that EPS at near-plasma concentration increases the intracellular levels of glutathione (GSH, which is important for protection against oxidative injury, through transcription regulation. Treatment of Schwann cells with EPS caused a dramatic increase in intracellular GSH levels. EPS increased the mRNA levels of γ-glutamylcysteine synthetase (γ-GCS, the enzyme catalyzing the first and rate-limiting step in de novo GSH synthesis. Nuclear factor erythroid 2-related factor 2 (Nrf2 is a key transcription factor that plays a central role in regulating the expression of γ-GCS. ELISA revealed that EPS increased nuclear Nrf2 levels. Knockdown of Nrf2 by siRNA suppressed the EPS-induced GSH biosynthesis. Furthermore, pretreatment with EPS reduced the cytotoxicity induced by H2O2, tert-butylhydroperoxide, 2,2'-azobis (2-amidinopropane dihydrochloride, and menadione, indicating that EPS plays a role in protecting against oxidative stress. This is the first study to show that EPS induces GSH biosynthesis via the activation of Nrf2. We suggest that EPS has new beneficial properties that may prevent the development and progression of disorders caused by oxidative stress.

  6. A history of plant biotechnology: from the Cell Theory of Schleiden and Schwann to biotech crops.

    Science.gov (United States)

    Vasil, Indra K

    2008-09-01

    Plant biotechnology is founded on the principles of cellular totipotency and genetic transformation, which can be traced back to the Cell Theory of Matthias Jakob Schleiden and Theodor Schwann, and the discovery of genetic transformation in bacteria by Frederick Griffith, respectively. On the 25th anniversary of the genetic transformation of plants, this review provides a historical account of the evolution of the theoretical concepts and experimental strategies that led to the production and commercialization of biotech (transformed or transgenic) plants expressing many useful genes, and emphasizes the beneficial effects of plant biotechnology on food security, human health, the environment, and conservation of biodiversity. In so doing, it celebrates and pays tribute to the contributions of scores of scientists who laid the foundation of modern plant biotechnology by their bold and unconventional thinking and experimentation. It highlights also the many important lessons to be learnt from the fascinating history of plant biotechnology, the significance of history in science teaching and research, and warns against the danger of the growing trends of ignoring history and historical illiteracy.

  7. Geometrical versus Random β-TCP Scaffolds: Exploring the Effects on Schwann Cell Growth and Behavior.

    Directory of Open Access Journals (Sweden)

    Lauren Sweet

    Full Text Available Numerous studies have demonstrated that Schwann cells (SCs play a role in nerve regeneration; however, their role in innervating a bioceramic scaffold for potential application in bone regeneration is still unknown. Here we report the cell growth and functional behavior of SCs on β-tricalcium phosphate (β-TCP scaffolds arranged in 3D printed-lattice (P-β-TCP and randomly-porous, template-casted (N-β-TCP structures. Our results indicate that SCs proliferated well and expressed the phenotypic markers p75LNGFR and the S100-β subunit of SCs as well as displayed growth morphology on both scaffolds, but SCs showed spindle-shaped morphology with a significant degree of SCs alignment on the P-β-TCP scaffolds, seen to a lesser degree in the N-β-TCP scaffold. The gene expressions of nerve growth factor (β-ngf, neutrophin-3 (nt-3, platelet-derived growth factor (pdgf-bb, and vascular endothelial growth factor (vegf-a were higher at day 7 than at day 14. While no significant differences in protein secretion were measured between these last two time points, the scaffolds promoted the protein secretion at day 3 compared to that on the cell culture plates. These results together imply that the β-TCP scaffolds can support SC cell growth and that the 3D-printed scaffold appeared to significantly promote the alignment of SCs along the struts. Further studies are needed to investigate the early and late stage relationship between gene expression and protein secretion of SCs on the scaffolds with refined characteristics, thus better exploring the potential of SCs to support vascularization and innervation in synthetic bone grafts.

  8. Promoting survival, migration, and integration of transplanted Schwann cells by over-expressing polysialic acid.

    Science.gov (United States)

    Luo, Juan; Bo, Xuenong; Wu, Dongsheng; Yeh, John; Richardson, Peter M; Zhang, Yi

    2011-03-01

    The poor survival and migration of transplanted Schwann cells (SCs) are major drawbacks for their clinical application in cell therapy for neurotrauma. To overcome such drawbacks we genetically modified SCs to over-express polysialic acid (PSA) by lentiviral delivery of polysialyltransferase (PST) to study whether over-expression of PSA could enhance their survival, migration, and integration when transplanted into the spinal cord. It was found that more PSA-expressing SCs (PST/SCs) survived than GFP-expressing SCs (GFP/SCs) after transplantation, although cell loss was still quite significant. PSA expression did not enhance the motility of transplanted SCs in uninjured spinal cord. However, in a spinal cord crush injury model PST/SCs transplanted caudal to the lesion showed that increased number of PST/SCs migrated to the injury site compared with that of GFP/SCs. Induced expression of PSA in spinal cord can further facilitate the infiltration of PST/SCs into the lesion site. PST/SCs were also shown to intermingle well with host spinal cells while GFP/SCs formed boundaries with host tissue. This was confirmed by an in vitro confrontation assay showing that more PST/SCs crossed over to astrocyte territory than GFP/SCs. Furthermore, PST/SCs induced much less expression of glial fibrillary acidic protein and chondroitin sulfate proteoglycan in the surrounding tissues than GFP/SCs, indicating that expression of PSA on SCs do not cause significant stress response of astrocytes. These results demonstrate that expression of PSA on SCs significantly changes their biological properties and makes them more feasible for neural repair after neurotrauma. Copyright © 2010 Wiley-Liss, Inc.

  9. Geometrical versus Random β-TCP Scaffolds: Exploring the Effects on Schwann Cell Growth and Behavior.

    Science.gov (United States)

    Sweet, Lauren; Kang, Yunqing; Czisch, Christopher; Witek, Lukasz; Shi, Yang; Smay, Jim; Plant, Giles W; Yang, Yunzhi

    2015-01-01

    Numerous studies have demonstrated that Schwann cells (SCs) play a role in nerve regeneration; however, their role in innervating a bioceramic scaffold for potential application in bone regeneration is still unknown. Here we report the cell growth and functional behavior of SCs on β-tricalcium phosphate (β-TCP) scaffolds arranged in 3D printed-lattice (P-β-TCP) and randomly-porous, template-casted (N-β-TCP) structures. Our results indicate that SCs proliferated well and expressed the phenotypic markers p75LNGFR and the S100-β subunit of SCs as well as displayed growth morphology on both scaffolds, but SCs showed spindle-shaped morphology with a significant degree of SCs alignment on the P-β-TCP scaffolds, seen to a lesser degree in the N-β-TCP scaffold. The gene expressions of nerve growth factor (β-ngf), neutrophin-3 (nt-3), platelet-derived growth factor (pdgf-bb), and vascular endothelial growth factor (vegf-a) were higher at day 7 than at day 14. While no significant differences in protein secretion were measured between these last two time points, the scaffolds promoted the protein secretion at day 3 compared to that on the cell culture plates. These results together imply that the β-TCP scaffolds can support SC cell growth and that the 3D-printed scaffold appeared to significantly promote the alignment of SCs along the struts. Further studies are needed to investigate the early and late stage relationship between gene expression and protein secretion of SCs on the scaffolds with refined characteristics, thus better exploring the potential of SCs to support vascularization and innervation in synthetic bone grafts.

  10. Transplantation of autologous Schwann cells for the repair of segmental peripheral nerve defects.

    Science.gov (United States)

    Hood, Brian; Levene, Howard B; Levi, Allan D

    2009-02-01

    Peripheral nerve injuries are a source of chronic disability. Incomplete recovery from such injuries results in motor and sensory dysfunction and the potential for the development of chronic pain. The repair of human peripheral nerve injuries with traditional surgical techniques has limited success, particularly when a damaged nerve segment needs to be replaced. An injury to a long segment of peripheral nerve is often repaired using autologous grafting of "noncritical" sensory nerve. Although extensive axonal regeneration can be observed extending into these grafts, recovery of function may be absent or incomplete if the axons fail to reach their intended target. The goal of this review was to summarize the progress that has occurred in developing an artificial neural prosthesis consisting of autologous Schwann cells (SCs), and to detail future directions required in translating this promising therapy to the clinic. In the authors' laboratory, methods are being explored to combine autologous SCs isolated using cell culture techniques with axon guidance channel (AGC) technology to develop the potential to repair critical gap length lesions within the peripheral nervous system. To test the clinical efficacy of such constructs, it is critically important to characterize the fate of the transplanted SCs with regard to cell survival, migration, differentiation, and myelin production. The authors sought to determine whether the use of SC-filled channels is superior or equivalent to strategies that are currently used clinically (for example, autologous nerve grafts). Finally, although many nerve repair paradigms demonstrate evidence of regeneration within the AGC, the authors further sought to determine if the regeneration observed was physiologically relevant by including electrophysiological, behavioral, and pain assessments. If successful, the development of this reparative approach will bring together techniques that are readily available for clinical use and should

  11. A systematic evaluation of Schwann cell injection into acellular cold-preserved nerve grafts.

    Science.gov (United States)

    Jesuraj, Nithya J; Santosa, Katherine B; Newton, Piyaraj; Liu, Z; Hunter, Daniel A; Mackinnon, Susan E; Sakiyama-Elbert, Shelly E; Johnson, Philip J

    2011-04-30

    Peripheral nerve regeneration after injury depends on environmental cues and trophic support. Schwann cells (SCs) secrete trophic factors that promote neuronal survival and help guide axons during regeneration. The addition of SCs to acellular nerve grafts is a promising strategy for enhancing peripheral nerve regeneration; however, inconsistencies in seeding parameters have led to varying results. The current work sought to establish a systematic approach to seeding SCs in cold-preserved acellular nerve grafts. Studies were undertaken to (1) determine the needle gauge for optimal cell survival and minimal epineurial disruption during injection, (2) track the seeded SCs using a commercially available dye, and (3) evaluate the seeding efficiency of SCs in nerve grafts. It was determined that seeding with a 27-gauge needle resulted in the highest viability of SCs with the least damage to the epineurium. In addition, Qtracker(®) dye, a commercially available quantum dot nanocrystal, was used to label SCs prior to transplantation, which allowed visualization of the seeded SCs in nerve grafts. Finally, stereological methods were used to evaluate the seeding efficiency of SCs in nerve grafts immediately after injection and following a 1- or 3-day in vitro incubation in SC growth media. Using a systematic approach, the best needle gauge and a suitable dye for SC visualization in acellular nerve grafts were identified. Seeding efficiency in these grafts was also determined. The findings will lead to improvements ability to assess injection of cells (including SCs) for use with acellular nerve grafts to promote nerve regeneration. Copyright © 2011 Elsevier B.V. All rights reserved.

  12. Embryonic stem cells and property rights.

    Science.gov (United States)

    Andersson, Anna-Karin M

    2011-06-01

    This article contributes to the current debate on human embryonic stem cell researchers' possible complicity in the destruction of human embryos and the relevance of such complicity for the issue of commodification of human embryos. I will discuss if, and to what extent, researchers who destroy human embryos, and researchers who merely use human embryos destroyed by others, have moral use rights, and/or moral property rights, in these embryos. I argue that the moral status of the human embryo, however justified, places few restrictions on the latter researchers' use of it, and property rights in it, once it is destroyed. I argue that the former researchers have no property rights in the destroyed embryo but use rights in it to the extent allowed by the legitimate owners of the destroyed embryo. I discuss the implications of this account for previous and current US federal law regulating human embryonic stem cell research.

  13. Changes in the Coding and Non-coding Transcriptome and DNA Methylome that Define the Schwann Cell Repair Phenotype after Nerve Injury.

    Science.gov (United States)

    Arthur-Farraj, Peter J; Morgan, Claire C; Adamowicz, Martyna; Gomez-Sanchez, Jose A; Fazal, Shaline V; Beucher, Anthony; Razzaghi, Bonnie; Mirsky, Rhona; Jessen, Kristjan R; Aitman, Timothy J

    2017-09-12

    Repair Schwann cells play a critical role in orchestrating nerve repair after injury, but the cellular and molecular processes that generate them are poorly understood. Here, we perform a combined whole-genome, coding and non-coding RNA and CpG methylation study following nerve injury. We show that genes involved in the epithelial-mesenchymal transition are enriched in repair cells, and we identify several long non-coding RNAs in Schwann cells. We demonstrate that the AP-1 transcription factor C-JUN regulates the expression of certain micro RNAs in repair Schwann cells, in particular miR-21 and miR-34. Surprisingly, unlike during development, changes in CpG methylation are limited in injury, restricted to specific locations, such as enhancer regions of Schwann cell-specific genes (e.g., Nedd4l), and close to local enrichment of AP-1 motifs. These genetic and epigenomic changes broaden our mechanistic understanding of the formation of repair Schwann cell during peripheral nervous system tissue repair. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  14. Embryonic stem cells in pig and cattle

    DEFF Research Database (Denmark)

    Maddox-Hyttel, Poul; Wolf, Xenia Asbæk; Rasmussen, Mikkel Aabech

    2007-01-01

    Porcine and bovine cell lines derived from the inner cell mass (ICM) or epiblasts of blastocysts have been maintained over extended periods of time and characterized by morphology, identification of some stem cell markers and, in few cases, by production of chimaeric offspring. However, germ line...... transmission in chimaeras has never been obtained. Due to this incomplete characterization of the cell lines, the expression embryonic stem (ES)-like cells is presently used in pig and cattle. The ICM or epiblast can be isolated from the blastocyst by whole blastocyst culture, mechanical isolation......, or immunosurgery, and they are generally cultured on feeder cells. The resulting ES-like cells may be differentiated in vivo by chimaera and teratoma formation or in vitro by embryoid body formation and monolayer induction. It is likely that more well characterized and stable porcine and bovine ES cell lines...

  15. Epigenetic control of embryonic stem cell fate

    DEFF Research Database (Denmark)

    Christophersen, Nicolaj Strøyer; Helin, Kristian

    2010-01-01

    Embryonic stem (ES) cells are derived from the inner cell mass of the preimplantation embryo and are pluripotent, as they are able to differentiate into all cell types of the adult organism. Once established, the pluripotent ES cells can be maintained under defined culture conditions, but can also...... be induced rapidly to differentiate. Maintaining this balance of stability versus plasticity is a challenge, and extensive studies in recent years have focused on understanding the contributions of transcription factors and epigenetic enzymes to the "stemness" properties of these cells. Identifying...... the molecular switches that regulate ES cell self-renewal versus differentiation can provide insights into the nature of the pluripotent state and enhance the potential use of these cells in therapeutic applications. Here, we review the latest models for how changes in chromatin methylation can modulate ES cell...

  16. Merlin status regulates p75(NTR) expression and apoptotic signaling in Schwann cells following nerve injury.

    Science.gov (United States)

    Ahmad, Iram; Fernando, Augusta; Gurgel, Richard; Jason Clark, J; Xu, Linjing; Hansen, Marlan R

    2015-10-01

    After nerve injury, Schwann cells (SCs) dedifferentiate, proliferate, and support axon regrowth. If axons fail to regenerate, denervated SCs eventually undergo apoptosis due, in part, to increased expression of the low-affinity neurotrophin receptor, p75(NTR). Merlin is the protein product of the NF2 tumor suppressor gene implicated in SC tumorigenesis. Here we explore the contribution of merlin to SC responses to nerve injury. We find that merlin becomes phosphorylated (growth permissive) in SCs following acute axotomy and following gradual neural degeneration in a deafness model, temporally correlated with increased p75(NTR) expression. p75(NTR) levels are elevated in P0SchΔ39-121 transgenic mice that harbor an Nf2 mutation in SCs relative to wild-type mice before axotomy and remain elevated for a longer period of time following injury. Replacement of wild-type, but not phospho-mimetic (S518D), merlin isoforms suppresses p75(NTR) expression in primary human schwannoma cultures which otherwise lack functional merlin. Despite elevated levels of p75(NTR), SC apoptosis following axotomy is blunted in P0SchΔ39-121 mice relative to wild-type mice suggesting that loss of functional merlin contributes to SC resistance to apoptosis. Further, cultured SCs from mice with a tamoxifen-inducible knock-out of Nf2 confirm that SCs lacking functional merlin are less sensitive to p75(NTR)-mediated cell death. Taken together these results point to a model whereby loss of axonal contact following nerve injury results in merlin phosphorylation leading to increased p75(NTR) expression. Further, they demonstrate that merlin facilitates p75(NTR)-mediated apoptosis in SCs helping to explain how neoplastic SCs that lack functional merlin survive long-term in the absence of axonal contact. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Merlin status regulates p75NTR expression and apoptotic signaling in Schwann cells following nerve injury

    Science.gov (United States)

    Ahmad, Iram; Fernando, Augusta; Gurgel, Richard; Clark, J. Jason; Xu, Linjing; Hansen, Marlan R.

    2015-01-01

    After nerve injury, Schwann cells (SCs) dedifferentiate, proliferate, and support axon regrowth. If axons fail to regenerate, denervated SCs eventually undergo apoptosis due, in part, to increased expression of the low-affinity neurotrophin receptor, p75NTR. Merlin is the protein product of the NF2 tumor suppressor gene implicated in SC tumorigenesis. Here we explore the contribution of merlin to SCs responses to nerve injury. We find that merlin becomes phosphorylated (growth permissive) in SCs following acute axotomy and following gradual neural degeneration in a deafness model, temporally correlated with increased p75NTR expression. p75NTR levels are elevated in P0SchΔ39-121 transgenic mice that harbor a Nf2 mutation in SCs relative to wild-type mice before axotomy and remain elevated for a longer period of time following injury. Replacement of wild-type, but not phosphomimetic (S518D), merlin isoforms suppresses p75NTR expression in primary human schwannoma cultures which otherwise lack functional merlin. Despite elevated levels of p75NTR, SC apoptosis following axotomy is blunted in P0SchΔ39-121 mice relative to wild-type mice suggesting that loss of functional merlin contributes to SC resistance to apoptosis. Further, cultured SCs from mice with a tamoxifen-inducible knock-out of Nf2 confirm that SCs lacking functional merlin are less-sensitive to p75NTR-mediated cell death. Taken together these results point to a model whereby loss of axonal contact following nerve injury results in merlin phosphorylation leading to increased p75NTR expression. Further, they demonstrate that merlin facilitates p75NTR-mediated apoptosis in SCs helping to explain how neoplastic SCs that lack functional merlin survive long-term in the absence of axonal contact. PMID:26057084

  18. Stepwise development of hematopoietic stem cells from embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Kenji Matsumoto

    Full Text Available The cellular ontogeny of hematopoietic stem cells (HSCs remains poorly understood because their isolation from and their identification in early developing small embryos are difficult. We attempted to dissect early developmental stages of HSCs using an in vitro mouse embryonic stem cell (ESC differentiation system combined with inducible HOXB4 expression. Here we report the identification of pre-HSCs and an embryonic type of HSCs (embryonic HSCs as intermediate cells between ESCs and HSCs. Both pre-HSCs and embryonic HSCs were isolated by their c-Kit(+CD41(+CD45(- phenotype. Pre-HSCs did not engraft in irradiated adult mice. After co-culture with OP9 stromal cells and conditional expression of HOXB4, pre-HSCs gave rise to embryonic HSCs capable of engraftment and long-term reconstitution in irradiated adult mice. Blast colony assays revealed that most hemangioblast activity was detected apart from the pre-HSC population, implying the early divergence of pre-HSCs from hemangioblasts. Gene expression profiling suggests that a particular set of transcripts closely associated with adult HSCs is involved in the transition of pre-HSC to embryonic HSCs. We propose an HSC developmental model in which pre-HSCs and embryonic HSCs sequentially give rise to adult types of HSCs in a stepwise manner.

  19. Netrin-1 induces the migration of Schwann cells via p38 MAPK and PI3K-Akt signaling pathway mediated by the UNC5B receptor

    Energy Technology Data Exchange (ETDEWEB)

    Lv, Jianwei [General Hospital of Tianjin Medical University, No. 154, Anshan Road, Heping District, Tianjin 300052 (China); Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin 300050 (China); Sun, Xiaolei; Ma, Jianxiong [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin 300050 (China); Ma, Xinlong, E-mail: gengxiao502@163.com [General Hospital of Tianjin Medical University, No. 154, Anshan Road, Heping District, Tianjin 300052 (China); Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin 300050 (China); Zhang, Yang; Li, Fengbo; Li, Yanjun; Zhao, Zhihu [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin 300050 (China)

    2015-08-14

    Schwann cells (SCs) play an essentially supportive role in the regeneration of injured peripheral nerve system (PNS). As Netrin-1 is crucial for the normal development of nervous system (NS) and can direct the process of damaged PNS regeneration, our study was designed to determine the role of Netrin-1 in RSC96 Schwann cells (an immortalized rat Schwann cell line) proliferation and migration. Our studies demonstrated that Netrin-1 had no effect on RSC96 cells proliferation, while significantly promoted RSC96 cells migration. The Netrin-1-induced RSC96 cells migration was significantly attenuated by inhibition of p38 and PI3K through pretreatment with SB203580 and LY294002 respectively, but not inhibition of MEK1/2 and JNK by U0126-EtOH and SP600125 individually. Treatment with Netrin-1 enhanced the phosphorylation of p38 and Akt. QRT-PCR indicated that Netrin-1 and only its receptors Unc5a, Unc5b and Neogenin were expressed in RSC96 cells, among which Unc5b expressed the most. And UNC5B protein was significantly increased after stimulated by Netrin-1. In conclusion, we show here that Netrin-1-enhanced SCs migration is mediated by activating p38 MAPK and PI3K-Akt signal cascades via receptor UNC5B, which suggests that Netrin-1 could serve as a new therapeutic strategy and has potential application value for PNS regeneration. - Highlights: • Netrin-1 attracts RSC96 Schwann cells migration in a dose dependent manner. • Netrin-1 induced Schwann cells migration is p38 and PI3K-Akt signaling dependent. • UNC5B may be dominant receptor mediating Netrin-1′ effect on RSC96 cells motility. • Netrin-1 may promote peripheral nerve repair by enhancing Schwann cells motility.

  20. Cultured Schwann cells assemble normal-appearing basal lamina only when they ensheathe axons.

    Science.gov (United States)

    Clark, M B; Bunge, M B

    1989-06-01

    Previous work demonstrated that Schwann cells (SCs) must interact with nerve cells (NCs) in order to generate their basal lamina (BL) in culture (M. B. Bunge, A. K. Williams, and P. M. Wood, 1982, Dev. Biol. 92, 449-460). The present study was undertaken to determine if this interaction requires proximity of NCs to SCs. Coverslips carrying isolated SCs were placed into culture dishes containing normally contacting SCs + NCs, NCs alone, or SCs alone and were maintained in these dishes for 3-4 weeks in medium known to foster the differentiation of axon-related SCs (BL formation, myelination). The SCs on the coverslip were not allowed to contact the cells in the culture dish. In other experiments, SCs isolated on coverslips were simply cultured in medium conditioned by contacting SCs + NCs, NCs alone, or SCs alone. The accumulation of BL components was monitored by light microscopic immunocytochemistry and the assembly of BL structure assessed by electron microscopy. When SCs were cocultured with but not contacted by neurons, immunostaining for BL constituents revealed a patchy deposition of material in sharp contrast to the linear deposition observed on axon-related SCs. Electron microscopy of these isolated SCs revealed short segments of BL, strands or clumps of BL-like material extending away from the cell surface, and accumulation of this material between cells. A greater number of isolated SCs were immunostained when grown with contacting SCs + NCs than with NCs or SCs. The conditioned medium experiments yielded similar results; only patchy BL was observed and more immunostaining was detected on isolated SCs when the medium had been conditioned by contacting SCs + NCs than by NCs alone or SCs alone. Immunostaining was less overall in the conditioned medium experiments than in the cell coculture work. In addition, standard SC + NC cultures grown in differentiation-supporting medium were studied by electron microscopy. SCs that were not contacted by axons but were

  1. Endothelial cells derived from human embryonic stem cells

    Science.gov (United States)

    Levenberg, Shulamit; Golub, Justin S.; Amit, Michal; Itskovitz-Eldor, Joseph; Langer, Robert

    2002-04-01

    Human embryonic stem cells have the potential to differentiate into various cell types and, thus, may be useful as a source of cells for transplantation or tissue engineering. We describe here the differentiation steps of human embryonic stem cells into endothelial cells forming vascular-like structures. The human embryonic-derived endothelial cells were isolated by using platelet endothelial cell-adhesion molecule-1 (PECAM1) antibodies, their behavior was characterized in vitro and in vivo, and their potential in tissue engineering was examined. We show that the isolated embryonic PECAM1+ cells, grown in culture, display characteristics similar to vessel endothelium. The cells express endothelial cell markers in a pattern similar to human umbilical vein endothelial cells, their junctions are correctly organized, and they have high metabolism of acetylated low-density lipoprotein. In addition, the cells are able to differentiate and form tube-like structures when cultured on matrigel. In vivo, when transplanted into SCID mice, the cells appeared to form microvessels containing mouse blood cells. With further studies, these cells could provide a source of human endothelial cells that could be beneficial for potential applications such as engineering new blood vessels, endothelial cell transplantation into the heart for myocardial regeneration, and induction of angiogenesis for treatment of regional ischemia.

  2. Intestinal lineage commitment of embryonic stem cells.

    Science.gov (United States)

    Cao, Li; Gibson, Jason D; Miyamoto, Shingo; Sail, Vibhavari; Verma, Rajeev; Rosenberg, Daniel W; Nelson, Craig E; Giardina, Charles

    2011-01-01

    Generating lineage-committed intestinal stem cells from embryonic stem cells (ESCs) could provide a tractable experimental system for understanding intestinal differentiation pathways and may ultimately provide cells for regenerating damaged intestinal tissue. We tested a two-step differentiation procedure in which ESCs were first cultured with activin A to favor formation of definitive endoderm, and then treated with fibroblast-conditioned medium with or without Wnt3A. The definitive endoderm expressed a number of genes associated with gut-tube development through mouse embryonic day 8.5 (Sox17, Foxa2, and Gata4 expressed and Id2 silent). The intestinal stem cell marker Lgr5 gene was also activated in the endodermal cells, whereas the Msi1, Ephb2, and Dcamkl1 intestinal stem cell markers were not. Exposure of the endoderm to fibroblast-conditioned medium with Wnt3A resulted in the activation of Id2, the remaining intestinal stem cell markers and the later gut markers Cdx2, Fabp2, and Muc2. Interestingly, genes associated with distal gut-associated mesoderm (Foxf2, Hlx, and Hoxd8) were also simulated by Wnt3A. The two-step differentiation protocol generated gut bodies with crypt-like structures that included regions of Lgr5-expressing proliferating cells and regions of cell differentiation. These gut bodies also had a smooth muscle component and some underwent peristaltic movement. The ability of the definitive endoderm to differentiate into intestinal epithelium was supported by the vivo engraftment of these cells into mouse colonic mucosa. These findings demonstrate that definitive endoderm derived from ESCs can carry out intestinal cell differentiation pathways and may provide cells to restore damaged intestinal tissue. Copyright © 2010 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

  3. Schwann cell-derived Apolipoprotein D controls the dynamics of post-injury myelin recognition and degradation

    Directory of Open Access Journals (Sweden)

    Nadia eGarcía-Mateo

    2014-11-01

    Full Text Available Management of lipids, particularly signaling lipids that control neuroinflammation, is crucial for the regeneration capability of a damaged nervous system. Knowledge of pro- and anti-inflammatory signals after nervous system injury is extensive, most of them being proteins acting through well-known receptors and intracellular cascades. However, the role of lipid binding extracellular proteins able to modify the fate of lipids released after injury is not well understood.Apolipoprotein D (ApoD is an extracellular lipid binding protein of the Lipocalin family induced upon nervous system injury. Our previous study shows that axon regeneration is delayed without ApoD, and suggests its participation in early events during Wallerian degeneration. Here we demonstrate that ApoD is expressed by myelinating and non-myelinating Schwann cells and is induced early upon nerve injury. We show that ApoD, known to bind arachidonic acid (AA, also interacts with lysophosphatidylcholine (LPC in vitro. We use an in vivo model of nerve crush injury, a nerve explant injury model, and cultured macrophages exposed to purified myelin, to uncover that: (i ApoD regulates denervated Schwann cell-macrophage signaling, dampening MCP1- and Tnf-dependent macrophage recruitment and activation upon injury; (ii ApoD controls the over-expression of the phagocytosis activator Galectin-3 by infiltrated macrophages; (iii ApoD controls the basal and injury-triggered levels of LPC and AA; (iv ApoD modifies the dynamics of myelin-macrophage interaction, favoring the initiation of phagocytosis and promoting myelin degradation.Regulation of macrophage behaviour by Schwann-derived ApoD is therefore a key mechanism conditioning nerve injury resolution. These results place ApoD as a lipid binding protein controlling the signals exchanged between glia, neurons and blood-borne cells during nerve recovery after injury, and open the possibility for a therapeutic use of ApoD as a regeneration

  4. Association of Myosin Va and Schwann cells-derived RNA in mammal myelinated axons, analyzed by immunocytochemistry and confocal FRET microscopy.

    Science.gov (United States)

    Canclini, Lucía; Wallrabe, Horst; Di Paolo, Andrés; Kun, Alejandra; Calliari, Aldo; Sotelo-Silveira, José Roberto; Sotelo, José Roberto

    2014-03-15

    Evidence from multiple sources supports the hypothesis that Schwann cells in the peripheral nervous system transfer messenger RNA and ribosomes to the axons they ensheath. Several technical and methodological difficulties exist for investigators to unravel this process in myelinated axons - a complex two-cell unit. We present an experimental design to demonstrate that newly synthesized RNA is transferred from Schwann cells to axons in association with Myosin Va. The use of quantitative confocal FRET microscopy to track newly-synthesized RNA and determine the molecular association with Myosin Va, is described in detail. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Macrophage-derived microvesicles promote proliferation and migration of Schwann cell on peripheral nerve repair

    Energy Technology Data Exchange (ETDEWEB)

    Zhan, Chuan, E-mail: zhchuansy@163.com; Ma, Cheng-bin; Yuan, Hong-mou; Cao, Bao-yuan; Zhu, Jia-jun

    2015-12-04

    Background: Macrophages have been implicated in peripheral nerve regeneration. However, whether macrophages-derived microvesicles (MVs) are involved in this process remains unknown. In the present study, the effects of macrophages-derived MVs on proliferation and migration of Schwann cells (SCs) were evaluated in both in vitro and in vivo. Methods: Human monocytic leukaemia cell line (THP-1) was successfully driven to M1 and M2 phenotypes by delivery of either IFN-γ or IL-4, respectively. SCs incubated with M1 or M2 macrophages-derived MVs, the cell migration and proliferation were assessed, and expression levels of nerve growth factor (NGF) and Laminin were measured. A rat model of sciatic nerve was established and the effects of macrophages-derived MVs on nerve regeneration were investigated. Results: M2-derived MVs elevated migration, proliferation, NFG and Laminin protein levels of SCs compared with M1-or M0-derived MVs. The relative expression levels of miR-223 were also increased in M2 macrophages and M2-derived MVs. Transfected M2 macrophages with miR-223 inhibitor then co-incubated with SCs, an inhibition of cell migration and proliferation and a down-regulated levels of NFG and Laminin protein expression were observed. In vivo, M2-derived MVs significantly increased the infiltration and axon number of SCs. Conclusion: M2-derived MVs promoted proliferation and migration of SCs in vitro and in vivo, which provided a therapeutic strategy for nerve regeneration. - Highlights: • M2 macrophages-derived MVs elevated migration and proliferation of SCs. • M2 macrophages-derived MVs up-regulated NFG and Laminin expression of SCs. • MiR-223 expression was increased in M2 macrophages-derived MVs. • MiR-223 inhibitor reduced migration and proliferation of SCs co-incubated with MVs. • MiR-223 inhibitor down-regulated NFG and Laminin levels of SCs co-incubated with MVs.

  6. Enhanced Schwann cell attachment and alignment using one-pot "dual click" GRGDS and YIGSR derivatized nanofibers.

    Science.gov (United States)

    Zheng, Jukuan; Kontoveros, Dimitria; Lin, Fei; Hua, Geng; Reneker, Darrell H; Becker, Matthew L; Willits, Rebecca K

    2015-01-12

    Using metal-free click chemistry and oxime condensation methodologies, GRGDS and YIGSR peptides were coupled to random and aligned degradable nanofiber networks postelectrospinning in a one-pot reaction. The bound peptides are bioactive, as demonstrated by Schwann cell attachment and proliferation, and the inclusion of YIGSR with GRGDS alters the expression of the receptor for YIGSR. Additionally, aligned nanofibers act as a potential guidance cue by increasing the aspect ratio and aligning the actin filaments, which suggest that peptide-functionalized scaffolds would be useful to direct SCs for peripheral nerve regeneration.

  7. Human embryonic stem cells: preclinical perspectives

    Directory of Open Access Journals (Sweden)

    Sarda Kanchan

    2008-01-01

    Full Text Available Abstract Human embryonic stem cells (hESCs have been extensively discussed in public and scientific communities for their potential in treating diseases and injuries. However, not much has been achieved in turning them into safe therapeutic agents. The hurdles in transforming hESCs to therapies start right with the way these cells are derived and maintained in the laboratory, and goes up-to clinical complications related to need for patient specific cell lines, gender specific aspects, age of the cells, and several post transplantation uncertainties. The different types of cells derived through directed differentiation of hESC and used successfully in animal disease and injury models are described briefly. This review gives a brief outlook on the present and the future of hESC based therapies, and talks about the technological advances required for a safe transition from laboratory to clinic.

  8. Schwann cell expressed Nogo-B modulates axonal branching of adult sensory neurons through the Nogo-B receptor NgBR

    Directory of Open Access Journals (Sweden)

    Christoph eEckharter

    2015-11-01

    Full Text Available In contrast to the central nervous system (CNS nerve fibers do regenerate in the peripheral nervous system (PNS although in a clinically unsatisfying manner. A major problem is excessive sprouting of regenerating axons which results in aberrant reinnervation of target tissue and impaired functional recovery. In the CNS, the reticulon protein Nogo-A has been identified as a prominent oligodendrocyte expressed inhibitor of long-distance growth of regenerating axons. We show here that the related isoform Nogo-B is abundantly expressed in Schwann cells in the PNS. Other than Nogo-A in oligodendrocytes, Nogo-B does not localize to the myelin sheath but is detected in the ER and the plasma membrane of Schwann cells. Adult sensory neurons that are cultured on nogo-a/b deficient Schwann cells form significantly fewer axonal branches versus those on wildtype Schwann cells, while their maximal axonal extension is unaffected. We demonstrate that this effect of Nogo-B on neuronal morphology is restricted to undifferentiated Schwann cells and is mediated by direct physical contact between these two cell types. Moreover, we show that blocking the Nogo-B specific receptor NgBR, which we find expressed on sensory neurons and to interact with Schwann cell expressed Nogo-B, produces the same branching phenotype as observed after deletion of Nogo-B. These data provide evidence for a novel function of the nogo gene that is implemented by the Nogo-B isoform. The remarkably specific effects of Nogo-B/ NgBR on axonal branching, while leaving axonal extension unaffected, are of potential clinical relevance in the context of excessive axonal sprouting after peripheral nerve injury.

  9. Mapping the stem cell state: eight novel human embryonic stem and embryonal carcinoma cell antibodies

    DEFF Research Database (Denmark)

    Wright, A; Andrews, N; Bardsley, K

    2011-01-01

    The antigenic profile of human embryonic stem (ES) and embryonal carcinoma (EC) cells has served as a key element of their characterization, with a common panel of surface and intracellular markers now widely used. Such markers have been used to identify cells within the 'undifferentiated state...... of reactivity for all antibodies against both ES and EC cells, suggesting that these markers will afford recognition of unique sub-states within the undifferentiated stem cell compartment....... and EC cells, and herein describe their characterization. The reactivity of these antibodies against a range of cell lines is reported, as well as their developmental regulation, basic biochemistry and reactivity in immunohistochemistry of testicular germ cell tumours. Our data reveal a range...

  10. Requirement of cAMP signaling for Schwann cell differentiation restricts the onset of myelination.

    Directory of Open Access Journals (Sweden)

    Ketty Bacallao

    Full Text Available Isolated Schwann cells (SCs respond to cAMP elevation by adopting a differentiated post-mitotic state that exhibits high levels of Krox-20, a transcriptional enhancer of myelination, and mature SC markers such as the myelin lipid galactocerebroside (O1. To address how cAMP controls myelination, we performed a series of cell culture experiments which compared the differentiating responses of isolated and axon-related SCs to cAMP analogs and ascorbate, a known inducer of axon ensheathment, basal lamina formation and myelination. In axon-related SCs, cAMP induced the expression of Krox-20 and O1 without a concomitant increase in the expression of myelin basic protein (MBP and without promoting axon ensheathment, collagen synthesis or basal lamina assembly. When cAMP was provided together with ascorbate, a dramatic enhancement of MBP expression occurred, indicating that cAMP primes SCs to form myelin only under conditions supportive of basal lamina formation. Experiments using a combination of cell permeable cAMP analogs and type-selective adenylyl cyclase (AC agonists and antagonists revealed that selective transmembrane AC (tmAC activation with forskolin was not sufficient for full SC differentiation and that the attainment of an O1 positive state also relied on the activity of the soluble AC (sAC, a bicarbonate sensor that is insensitive to forskolin and GPCR activation. Pharmacological and immunological evidence indicated that SCs expressed sAC and that sAC activity was required for morphological differentiation and the expression of myelin markers such as O1 and protein zero. To conclude, our data indicates that cAMP did not directly drive myelination but rather the transition into an O1 positive state, which is perhaps the most critical cAMP-dependent rate limiting step for the onset of myelination. The temporally restricted role of cAMP in inducing differentiation independently of basal lamina formation provides a clear example of the

  11. Rat Nasal Respiratory Mucosa-Derived Ectomesenchymal Stem Cells Differentiate into Schwann-Like Cells Promoting the Differentiation of PC12 Cells and Forming Myelin In Vitro

    Directory of Open Access Journals (Sweden)

    Jian Zhang

    2015-01-01

    Full Text Available Schwann cell (SC transplantation as a cell-based therapy can enhance peripheral and central nerve repair experimentally, but it is limited by the donor site morbidity for clinical application. We investigated weather respiratory mucosa stem cells (REMSCs, a kind of ectomesenchymal stem cells (EMSCs, isolated from rat nasal septum can differentiate into functional Schwann-like cells (SC-like cells. REMSCs proliferated quickly in vitro and expressed the neural crest markers (nestin, vimentin, SOX10, and CD44. Treated with a mixture of glial growth factors for 7 days, REMSCs differentiated into SC-like cells. The differentiated REMSCs (dREMSCs exhibited a spindle-like morphology similar to SC cells. Immunocytochemical staining and Western blotting indicated that SC-like cells expressed the glial markers (GFAP, S100β, Galc, and P75 and CNPase. When cocultured with dREMSCs for 5 days, PC12 cells differentiated into mature neuron-like cells with long neurites. More importantly, dREMSCs could form myelin structures with the neurites of PC12 cells at 21 days in vitro. Our data indicated that REMSCs, a kind of EMSCs, could differentiate into SC-like cells and have the ability to promote the differentiation of PC12 cells and form myelin in vitro.

  12. Will embryonic stem cells change health policy?

    Science.gov (United States)

    Sage, William M

    2010-01-01

    Embryonic stem cells are actively debated in political and public policy arenas. However, the connections between stem cell innovation and overall health care policy are seldom elucidated. As with many controversial aspects of medical care, the stem cell debate bridges to a variety of social conversations beyond abortion. Some issues, such as translational medicine, commercialization, patient and public safety, health care spending, physician practice, and access to insurance and health care services, are core health policy concerns. Other issues, such as economic development, technologic progress, fiscal politics, and tort reform, are only indirectly related to the health care system but are frequently seen through a health care lens. These connections will help determine whether the stem cell debate reaches a resolution, and what that resolution might be.

  13. Gene delivery to rat and human Schwann cells and nerve segments : a comparison of AAV 1-9 and lentiviral vectors

    NARCIS (Netherlands)

    Hoyng, S A; De Winter, F; Gnavi, S; van Egmond, L; Attwell, C L; Tannemaat, M R; Verhaagen, J; Malessy, M J A

    2015-01-01

    Schwann cells (SCs) in an injured peripheral nerve form pathways for regenerating axons. Although these cells initially support regeneration, SCs lose their pro-regenerative properties following a prolonged period of denervation. Gene transfer to SC can enhance their therapeutic potential. In this

  14. Gene delivery to rat and human Schwann cells and nerve segments: a comparison of AAV 1-9 and lentiviral vectors

    NARCIS (Netherlands)

    Hoyng, S.A.; de Winter, F.; Gnavi, S.; van Egmond, L.; Attwell, C.L.; Tannemaat, M.R.; Verhaagen, J.; Malessy, M.J.A.

    2015-01-01

    Schwann cells (SCs) in an injured peripheral nerve form pathways for regenerating axons. Although these cells initially support regeneration, SCs lose their pro-regenerative properties following a prolonged period of denervation. Gene transfer to SC can enhance their therapeutic potential. In this

  15. Tonsil-Derived Mesenchymal Stem Cells Differentiate into a Schwann Cell Phenotype and Promote Peripheral Nerve Regeneration

    Science.gov (United States)

    Jung, Namhee; Park, Saeyoung; Choi, Yoonyoung; Park, Joo-Won; Hong, Young Bin; Park, Hyun Ho Choi; Yu, Yeonsil; Kwak, Geon; Kim, Han Su; Ryu, Kyung-Ha; Kim, Jae Kwang; Jo, Inho; Choi, Byung-Ok; Jung, Sung-Chul

    2016-01-01

    Schwann cells (SCs), which produce neurotropic factors and adhesive molecules, have been reported previously to contribute to structural support and guidance during axonal regeneration; therefore, they are potentially a crucial target in the restoration of injured nervous tissues. Autologous SC transplantation has been performed and has shown promising clinical results for treating nerve injuries and donor site morbidity, and insufficient production of the cells have been considered as a major issue. Here, we performed differentiation of tonsil-derived mesenchymal stem cells (T-MSCs) into SC-like cells (T-MSC-SCs), to evaluate T-MSC-SCs as an alternative to SCs. Using SC markers such as CAD19, GFAP, MBP, NGFR, S100B, and KROX20 during quantitative real-time PCR we detected the upregulation of NGFR, S100B, and KROX20 and the downregulation of CAD19 and MBP at the fully differentiated stage. Furthermore, we found myelination of axons when differentiated SCs were cocultured with mouse dorsal root ganglion neurons. The application of T-MSC-SCs to a mouse model of sciatic nerve injury produced marked improvements in gait and promoted regeneration of damaged nerves. Thus, the transplantation of human T-MSCs might be suitable for assisting in peripheral nerve regeneration. PMID:27834852

  16. Tonsil-Derived Mesenchymal Stem Cells Differentiate into a Schwann Cell Phenotype and Promote Peripheral Nerve Regeneration

    Directory of Open Access Journals (Sweden)

    Namhee Jung

    2016-11-01

    Full Text Available Schwann cells (SCs, which produce neurotropic factors and adhesive molecules, have been reported previously to contribute to structural support and guidance during axonal regeneration; therefore, they are potentially a crucial target in the restoration of injured nervous tissues. Autologous SC transplantation has been performed and has shown promising clinical results for treating nerve injuries and donor site morbidity, and insufficient production of the cells have been considered as a major issue. Here, we performed differentiation of tonsil-derived mesenchymal stem cells (T-MSCs into SC-like cells (T-MSC-SCs, to evaluate T-MSC-SCs as an alternative to SCs. Using SC markers such as CAD19, GFAP, MBP, NGFR, S100B, and KROX20 during quantitative real-time PCR we detected the upregulation of NGFR, S100B, and KROX20 and the downregulation of CAD19 and MBP at the fully differentiated stage. Furthermore, we found myelination of axons when differentiated SCs were cocultured with mouse dorsal root ganglion neurons. The application of T-MSC-SCs to a mouse model of sciatic nerve injury produced marked improvements in gait and promoted regeneration of damaged nerves. Thus, the transplantation of human T-MSCs might be suitable for assisting in peripheral nerve regeneration.

  17. Pathways in pluripotency and differentiation of embryonic cells

    NARCIS (Netherlands)

    du Puy, L.

    2010-01-01

    Pluripotency - the potential to differentiate into derivatives of the three embryonic germ layers endoderm, ectoderm and mesoderm - is the main characteristic of embryonic stem (ES) cells. ES cells are derived from the inner cell mass (ICM) of a pre-implantation blastocyst and can self-renew

  18. Reprogramming diminishes retention of Mycobacterium leprae in Schwann cells and elevates bacterial transfer property to fibroblasts [v2; ref status: indexed, http://f1000r.es/280

    Directory of Open Access Journals (Sweden)

    Toshihiro Masaki

    2013-11-01

    Full Text Available Background: Bacterial pathogens can manipulate or subvert host tissue cells to their advantage at different stages during infection, from initial colonization in primary host niches to dissemination. Recently, we have shown that Mycobacterium leprae (ML, the causative agent of human leprosy, reprogrammed its preferred host niche de-differentiated adult Schwann cells to progenitor/stem cell-like cells (pSLC which appear to facilitate bacterial spread. Here, we studied how this cell fate change influences bacterial retention and transfer properties of Schwann cells before and after reprogramming. Results: Using primary fibroblasts as bacterial recipient cells, we showed that non-reprogrammed Schwann cells, which preserve all Schwann cell lineage and differentiation markers, possess high bacterial retention capacity when co-cultured with skin fibroblasts; Schwann cells failed to transfer bacteria to fibroblasts at higher numbers even after co-culture for 5 days. In contrast, pSLCs, which are derived from the same Schwann cells but have lost Schwann cell lineage markers due to reprogramming, efficiently transferred bacteria to fibroblasts within 24 hours. Conclusions: ML-induced reprogramming converts lineage-committed Schwann cells with high bacterial retention capacity to a cell type with pSLC stage with effective bacterial transfer properties. We propose that such changes in cellular properties may be associated with the initial intracellular colonization, which requires long-term bacterial retention within Schwann cells, in order to spread the infection to other tissues, which entails efficient bacterial transfer capacity to cells like fibroblasts which are abundant in many tissues, thereby potentially maximizing bacterial dissemination. These data also suggest how pathogens could take advantage of multiple facets of host cell reprogramming according to their needs during infection.

  19. Reprogramming diminishes retention of Mycobacterium leprae in Schwann cells and elevates bacterial transfer property to fibroblasts [v3; ref status: indexed, http://f1000r.es/2ae

    Directory of Open Access Journals (Sweden)

    Toshihiro Masaki

    2013-11-01

    Full Text Available Background: Bacterial pathogens can manipulate or subvert host tissue cells to their advantage at different stages during infection, from initial colonization in primary host niches to dissemination. Recently, we have shown that Mycobacterium leprae (ML, the causative agent of human leprosy, reprogrammed its preferred host niche de-differentiated adult Schwann cells to progenitor/stem cell-like cells (pSLC which appear to facilitate bacterial spread. Here, we studied how this cell fate change influences bacterial retention and transfer properties of Schwann cells before and after reprogramming. Results: Using primary fibroblasts as bacterial recipient cells, we showed that non-reprogrammed Schwann cells, which preserve all Schwann cell lineage and differentiation markers, possess high bacterial retention capacity when co-cultured with skin fibroblasts; Schwann cells failed to transfer bacteria to fibroblasts at higher numbers even after co-culture for 5 days. In contrast, pSLCs, which are derived from the same Schwann cells but have lost Schwann cell lineage markers due to reprogramming, efficiently transferred bacteria to fibroblasts within 24 hours. Conclusions: ML-induced reprogramming converts lineage-committed Schwann cells with high bacterial retention capacity to a cell type with pSLC stage with effective bacterial transfer properties. We propose that such changes in cellular properties may be associated with the initial intracellular colonization, which requires long-term bacterial retention within Schwann cells, in order to spread the infection to other tissues, which entails efficient bacterial transfer capacity to cells like fibroblasts which are abundant in many tissues, thereby potentially maximizing bacterial dissemination. These data also suggest how pathogens could take advantage of multiple facets of host cell reprogramming according to their needs during infection.

  20. Hematopoietic cell differentiation from embryonic and induced pluripotent stem cells

    Science.gov (United States)

    2013-01-01

    Pluripotent stem cells, both embryonic stem cells and induced pluripotent stem cells, are undifferentiated cells that can self-renew and potentially differentiate into all hematopoietic lineages, such as hematopoietic stem cells (HSCs), hematopoietic progenitor cells and mature hematopoietic cells in the presence of a suitable culture system. Establishment of pluripotent stem cells provides a comprehensive model to study early hematopoietic development and has emerged as a powerful research tool to explore regenerative medicine. Nowadays, HSC transplantation and hematopoietic cell transfusion have successfully cured some patients, especially in malignant hematological diseases. Owing to a shortage of donors and a limited number of the cells, hematopoietic cell induction from pluripotent stem cells has been regarded as an alternative source of HSCs and mature hematopoietic cells for intended therapeutic purposes. Pluripotent stem cells are therefore extensively utilized to facilitate better understanding in hematopoietic development by recapitulating embryonic development in vivo, in which efficient strategies can be easily designed and deployed for the generation of hematopoietic lineages in vitro. We hereby review the current progress of hematopoietic cell induction from embryonic stem/induced pluripotent stem cells. PMID:23796405

  1. Human embryonic stem cells and embryonal carcinoma cells have overlapping and distinct metabolic signatures.

    Directory of Open Access Journals (Sweden)

    Raed Abu Dawud

    Full Text Available While human embryonic stem cells (hESCs and human embryonal carcinoma cells (hECCs have been studied extensively at the levels of the genome, transcriptome, proteome and epigenome our knowledge of their corresponding metabolomes is limited. Here, we present the metabolic signatures of hESCs and hESCs obtained by untargeted gas chromatography coupled to mass spectrometry (GC-MS. Whilst some metabolites are common to both cell types, representing the self-renewal and house-keeping signatures, others were either higher (e.g., octadecenoic acid, glycerol-3-phosphate, 4-hydroxyproline or lower (e.g., glutamic acid, mannitol, malic acid, GABA in hESCs (H9 compared to hECCs (NTERA2, these represent cell type specific signatures. Further, our combined results of GC-MS and microarray based gene expression profiling of undifferentiated and OCT4-depleted hESCs are consistent with the Warburg effect which is increased glycolysis in embryonic cells and tumor cells in the presence of O(2 while oxidative phosphorylation (OXPHOS is impaired or even shut down. RNAi-based OCT4 knock down mediated differentiation resulted in the activation of the poised OXPHOS machinery by expressing missing key proteins such as NDUFC1, UQCRB and COX, increase in TCA cycle activity and decreased lactate metabolism. These results shed light on the metabolite layer of pluripotent stem cells and could potentially establish novel metabolic markers of self renewal and pluripotency.

  2. Human embryonic stem cells and microenvironment

    Directory of Open Access Journals (Sweden)

    Banu İskender

    2014-09-01

    Full Text Available Human embryonic stem cells (hESCs possess a great potential in the field of regenerative medicine by their virtue of pluripotent potential with indefinite proliferation capabilities. They can self renew themselves and differentiate into three embryonic germ layers. Although they are conventionally grown on mitotically inactivated mouse feeder cells, there are in vitro culture systems utilizing feeder cells of human origin in order to prevent cross-species contamination. Recently established in vitro culture systems suggested that direct interaction with feeder cells is not necessary but rather attachment to a substrate is required to ensure long-term, efficient hESC culture in vitro. This substrate is usually composed of a mixture of extracellular matrix components representing in vivo natural niche. In hESC biology, the mechanism of interaction of hESCs with extracellular matrix molecules remained insufficiently explored area of research due to their transient nature of interaction with the in vivo niche. However, an in vitro culture system established using extracellular matrix molecules may provide a safer alternative to culture systems with feeder cells while paving the way to Good Manufacturing Practice-GMP production of hESCs for therapeutic purposes. Therefore, it is essential to study the interaction of extracellular matrix molecules with hESCs in order to standardize in vitro culture systems for large-scale production of hESCs in a less labor-intensive way. This would not only provide valuable information regarding the mechanisms that control pluripotency but also serve to dissect the molecular signaling pathways of directed differentiation for prospective therapeutic applications in the future. J Clin Exp Invest 2014; 5 (3: 486-495

  3. Uncoupled embryonic and extra-embryonic tissues compromise blastocyst development after somatic cell nuclear transfer.

    Directory of Open Access Journals (Sweden)

    Séverine A Degrelle

    Full Text Available Somatic cell nuclear transfer (SCNT is the most efficient cell reprogramming technique available, especially when working with bovine species. Although SCNT blastocysts performed equally well or better than controls in the weeks following embryo transfer at Day 7, elongation and gastrulation defects were observed prior to implantation. To understand the developmental implications of embryonic/extra-embryonic interactions, the morphological and molecular features of elongating and gastrulating tissues were analysed. At Day 18, 30 SCNT conceptuses were compared to 20 controls (AI and IVP: 10 conceptuses each; one-half of the SCNT conceptuses appeared normal while the other half showed signs of atypical elongation and gastrulation. SCNT was also associated with a high incidence of discordance in embryonic and extra-embryonic patterns, as evidenced by morphological and molecular "uncoupling". Elongation appeared to be secondarily affected; only 3 of 30 conceptuses had abnormally elongated shapes and there were very few differences in gene expression when they were compared to the controls. However, some of these differences could be linked to defects in microvilli formation or extracellular matrix composition and could thus impact extra-embryonic functions. In contrast to elongation, gastrulation stages included embryonic defects that likely affected the hypoblast, the epiblast, or the early stages of their differentiation. When taking into account SCNT conceptus somatic origin, i.e. the reprogramming efficiency of each bovine ear fibroblast (Low: 0029, Med: 7711, High: 5538, we found that embryonic abnormalities or severe embryonic/extra-embryonic uncoupling were more tightly correlated to embryo loss at implantation than were elongation defects. Alternatively, extra-embryonic differences between SCNT and control conceptuses at Day 18 were related to molecular plasticity (high efficiency/high plasticity and subsequent pregnancy loss. Finally

  4. The liberation of embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Kathryn Blair

    2011-04-01

    Full Text Available Mouse embryonic stem (ES cells are defined by their capacity to self-renew and their ability to differentiate into all adult tissues including the germ line. Along with efficient clonal propagation, these properties have made them an unparalleled tool for manipulation of the mouse genome. Traditionally, mouse ES (mES cells have been isolated and cultured in complex, poorly defined conditions that only permit efficient derivation from the 129 mouse strain; genuine ES cells have not been isolated from another species in these conditions. Recently, use of small molecule inhibitors of glycogen synthase kinase 3 (Gsk3 and the Fgf-MAPK signaling cascade has permitted efficient derivation of ES cells from all tested mouse strains. Subsequently, the first verified ES cells were established from a non-mouse species, Rattus norvegicus. Here, we summarize the advances in our understanding of the signaling pathways regulating mES cell self-renewal that led to the first derivation of rat ES cells and highlight the new opportunities presented for transgenic modeling on diverse genetic backgrounds. We also comment on the implications of this work for our understanding of pluripotent stem cells across mammalian species.

  5. Engineered neural tissue with Schwann cell differentiated human dental pulp stem cells: potential for peripheral nerve repair?

    Science.gov (United States)

    Sanen, Kathleen; Martens, Wendy; Georgiou, Melanie; Ameloot, Marcel; Lambrichts, Ivo; Phillips, James

    2017-01-04

    Despite the spontaneous regenerative capacity of the peripheral nervous system, large gap peripheral nerve injuries (PNIs) require bridging strategies. The limitations and suboptimal results obtained with autografts or hollow nerve conduits in the clinic urge the need for alternative treatments. Recently, we have described promising neuroregenerative capacities of Schwann cells derived from differentiated human dental pulp stem cells (d-hDPSCs) in vitro. Here, we extended the in vitro assays to show the pro-angiogenic effects of d-hDPSCs, such as enhanced endothelial cell proliferation, migration and differentiation. In addition, for the first time we evaluated the performance of d-hDPSCs in an in vivo rat model of PNI. Eight weeks after transplantation of NeuraWrap™ conduits filled with engineered neural tissue (EngNT) containing aligned d-hDPSCs in 15-mm rat sciatic nerve defects, immunohistochemistry and ultrastructural analysis revealed ingrowing neurites, myelinated nerve fibres and blood vessels along the construct. Although further research is required to optimize the delivery of this EngNT, our findings suggest that d-hDPSCs are able to exert a positive effect in the regeneration of nerve tissue in vivo. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  6. Human embryonic stem cells and patent protection

    Directory of Open Access Journals (Sweden)

    Radovanović Sanja M.

    2015-01-01

    Full Text Available Given the importance of biotechnological research in modern diagnostics and therapeutics, on the one hand, and stimulative function of a patent, on the other hand, this work deals with the question of the possibility of pa-tent protection of human embryonic stem cells. Taking into account that this is a biotechnological invention, the key question that this paper highlights is the interpretation of the provisions of their patentability. Namely, thanks to the advanced methods of isolation, purification and preparation for implementation, modern patent systems do not exclude a priori living organisms from patent protection. Therefore, the analysis of representative administrative decisions or court rulings sought to define the criteria that would be applied in order to give patent protection to a certain biotechnological invention (stem cells while others do not.

  7. LIF signal in mouse embryonic stem cells

    Science.gov (United States)

    Ohtsuka, Satoshi; Nakai-Futatsugi, Yoko; Niwa, Hitoshi

    2015-01-01

    Since the establishment of mouse embryonic stem cells (mESCs) in the 1980s, a number of important notions on the self-renewal of pluripotent stem cells in vitro have been found. In serum containing conventional culture, an exogenous cytokine, leukemia inhibitory factor (LIF), is absolutely essential for the maintenance of pluripotency. In contrast, in serum-free culture with simultaneous inhibition of Map-kinase and Gsk3 (so called 2i-culture), LIF is no longer required. However, recent findings also suggest that LIF may have a role not covered by the 2i for the maintenance of naïve pluripotency. These suggest that LIF functions for the maintenance of naïve pluripotency in a context dependent manner. We summarize how LIF-signal pathway is converged to maintain the naïve state of pluripotency. PMID:27127728

  8. Sam68 promotes Schwann cell proliferation by enhancing the PI3K/Akt pathway and acts on regeneration after sciatic nerve crush

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Weijie, E-mail: 459586768@qq.com; Liu, Yuxi, E-mail: 924013616@qq.com; Wang, Youhua, E-mail: wyouhua1516@163.com

    2016-05-13

    Sam68 (Src-associated in mitosis of 68 kD), a KH domain RNA-binding protein, is not only important in signaling transduction cascades, but crucial in a variety of cellular processes. Sam68 is reported to be involved in the phospoinositide3-kinase (PI3K) and nuclear factor-kappa B (NF-κB) signaling pathways, and it is closely associated with cell proliferation, RNA metabolism, and tumor progression. However, we know little about the role of Sam68 during peripheral nervous system injury and regeneration. In this study, we investigated the expression of Sam68 and its biological significances in sciatic nerve crush. Interestingly, we found Sam68 had a co-localization with S100 (Schwann cell marker). Moreover, after crush, Sam68 had a spatiotemporal protein expression, which was in parallel with proliferation cell nuclear antigen (PCNA). In vitro, we also observed increased expression of Sam68 during the process of TNF-α-induced Schwann cell proliferation model. Besides, flow cytometry analyses, CCK-8, and EDU were all performed with the purpose of investigating the role of Sam68 in the regulation of Schwann cell proliferation. Even more importantly, we discovered that Sam68 could enhance the phosphorylation of Akt while LY294002 (a PI3K inhibitor) obviously reversed Sam68-induced cell proliferation. Finally, we detected the variance during regeneration progress through the rat walk footprint test. In summary, all these evidences demonstrated that Sam68 might participate in Schwann cell proliferation partially via PI3K/Akt pathway and also regulate regeneration after sciatic nerve crush. -- Highlights: •The dynamic changes and location of Sam68 after sciatic nerve crush. •Sam68 promoted Schwann cell proliferation via PI3K/Akt pathway. •Sam68 modulated functional recovery after sciatic nerve crush.

  9. BD PuraMatrix peptide hydrogel as a culture system for human fetal Schwann cells in spinal cord regeneration.

    Science.gov (United States)

    Moradi, Fateme; Bahktiari, Mehrdad; Joghataei, Mohammad Taghi; Nobakht, Maliheh; Soleimani, Masoud; Hasanzadeh, Gholamreza; Fallah, Ali; Zarbakhsh, Sam; Hejazian, Leila Beigom; Shirmohammadi, Maryam; Maleki, Fatemeh

    2012-12-01

    BD PuraMatrix peptide hydrogel, a three-dimensional cell culture model of nanofiber scaffold derived from the self-assembling peptide RADA16, has been applied to regenerative tissue repair in order to develop novel nanomedicine systems. In this study with PuraMatrix, self-assembling nanofiber scaffold (SAPNS) and Schwann cells (SCs) were isolated from human fetal sciatic nerves, cultured within SAPNS, and then transplanted into the spinal cord after injury (SCI) in rats. First, the peptide nanofiber scaffold was evaluated via scanning electron microscopy and atomic force microscopy. With phase-contrast microscopy, the appearance of representative human fetal SCs encapsulated in PuraMatrix on days 3, 5, and 7 in 12-well plates was revealed. The Schwann cells in PuraMatrix were cultured for 2 days, and the SCs had active proliferative potential. Spinal cord injury was induced by placing a 35-g weight on the dura of T9-T10 segments for 15 min, followed by in vivo treatment with SAPNS and human fetal SCs (100,000 cells/10 μl/injection) grafted into spinal cord 7 days after SCI. After treatment, the recovery of motor function was assessed periodically using the Basso, Beattie, and Bresnahan scoring system. Eight weeks after grafting, animals were perfusion fixed, and the survival of implanted cells was analyzed with antibody recognizing SCs. Immunohistochemical analysis of grafted lumber segments at 8 weeks after grafting revealed reduced asterogliosis and considerably increased infiltration of endogenous S100(+) cells into the injury site, suggesting that PuraMatrix may play an important role in the repair observed after SAPNS and human fetal SC transplantation. Copyright © 2012 Wiley Periodicals, Inc.

  10. Tetramethylpyrazine protects Schwann cells from ischemia-like injury and increases cell survival in cold ischemic rat nerves

    Directory of Open Access Journals (Sweden)

    Ming-Ming Yang

    2015-03-01

    Full Text Available Tetramethylpyrazine (TMP, a major active ingredient of Ligusticum wallichi Franchat extract (a Chinese herb, exhibits neuroprotective properties in ischemia. In this study, we assessed its protective effects on Schwann cells (SCs by culturing them in the presence of oxygen glucose deprivation (OGD conditions and measuring cell survival in cold ischemic rat nerves. In the OGD-induced ischemic injury model of SCs, we demonstrated that TMP treatment not only reduced OGD-induced cell viability losses, cell death, and apoptosis of SCs in a dose-dependent manner, and inhibited LDH release, but also suppressed OGD-induced downregulation of Bcl-2 and upregulation of Bax and caspase-3, as well as inhibited the consequent activation of caspase-3. In the cold ischemic nerve model, we found that prolonged cold ischemic exposure for four weeks was markedly associated with the absence of SCs, a decrease in cell viability, and apoptosis in preserved nerve segments incubated in University of Wisconsin solution (UWS alone. However, TMP attenuated nerve segment damage by preserving SCs and antagonizing the decrease in nerve fiber viability and increase in TUNEL-positive cells in a dose-dependent manner. Collectively, our results indicate that TMP not only provides protective effects in an ischemia-like injury model of cultured rat SCs by regulating Bcl-2, Bax, and caspase-3, but also increases cell survival and suppresses apoptosis in the cold ischemic nerve model after prolonged ischemic exposure for four weeks. Therefore, TMP may be a novel and effective therapeutic strategy for preventing peripheral nervous system ischemic diseases and improving peripheral nerve storage.

  11. Sox2 in Embryonic Stem Cells and Lung Development

    NARCIS (Netherlands)

    C.G. Pardo (Cristina Gontan)

    2009-01-01

    markdownabstract__Abstract__ Sox2 is a fascinating transcription factor with multiple roles during embryonic development. In early embryonic development, Sox2 is one of the key transcription factors in the maintenance of the pluripotent status of the cells of the inner cell mass (ICM). Sox2 is

  12. A distal Schwann cell-specific enhancer mediates axonal regulation of the Oct-6 transcription factor during peripheral nerve development and regeneration.

    NARCIS (Netherlands)

    W.J. Mandemakers (Wim); R. Zwart (Ronald); M.M. Jaegle (Martine); E.T. Walbeehm (Erik); P. Visser (Pim); F.G. Grosveld (Frank); D. Meijer (Daniëlle)

    2000-01-01

    textabstractThe POU domain transcription factor Oct-6 is a major regulator of Schwann cell differentiation and myelination. During nerve development and regeneration, expression of Oct-6 is under the control of axonal signals. Identification of the cis-acting elements

  13. [Effect of Draconis Sanguis-containing serum on NGF, BDNF, CNTF, LNGFR, TrkA, GDNF, GAP-43 and NF-H expressions in Schwann cells].

    Science.gov (United States)

    Gu, Jin; He, Xin-rong; Han, Ya-liang

    2015-04-01

    To observe the effect of Draconis Sanguis-containing serum on the expressions of NGF, BDNF, CNTF, LNG-FR, TrkA, GDNF, GAP-43 and NF-H in Schwann cells, and investigate the possible mechanism of Draconis Sanguis to promote peripheral nerve regeneration. SD rats were randomly divided into 2 groups: the Draconis Sanguis group (orally administered with Draconis Sanguis-containing balm solution) and the blank group (equivoluminal balm) to prepare Draconis Sanguis-containing serum and blank control serum. Schwann cells were extracted from double sciatic nerves of three-day-old SD rats, divided into 2 groups: the Draconis Sanguis group and the blank control group, and respectively cultured with 10% Draconis Sanguis-containing serum or blank control serum. The mRNA expressions of NGF, BDNF, CNTF and other genes in Schwann cells were measured by RT-PCR analysis 48 hours later. Most of the Schwann cells were bipolar spindle and arranged shoulder to shoulder or end to end under the microscope and identified to be positive with the immunocytochemical method. To compare with the blank group, mRNA expressions of NGF, LNGFR, GDNF and GAP-43 significantly increased (P Sanguis may show effect in nerve regeneration by up-regulating mRNA expressions of NGF, LNGFR, GDNF and GAP-43 and down-regulating mRNA expressions of TrkA, BDNF and CNTF.

  14. Lysophospholipid receptors are differentially expressed in rat terminal Schwann cells, as revealed by a single cell rt-PCR and in situ hybridization.

    Science.gov (United States)

    Kobashi, Hiroaki; Yaoi, Takeshi; Oda, Ryo; Okajima, Seiichiro; Fujiwara, Hiroyoshi; Kubo, Toshikazu; Fushiki, Shinji

    2006-04-22

    Terminal Schwann cells (TSCs) that cover motor neuron terminals, are known to play an important role in maintaining neuromuscular junctions, as well as in the repair process after nerve injury. However, the molecular characteristics of TSCs remain unknown, because of the difficulties in analyzing them due to their paucity. By using our previously reported method of selectively and efficiently collecting TSCs, we have analyzed the difference in expression patterns of lysophospholipid (LPL) receptor genes (LPA1, LPA2, LPA3, S1P1, S1P2, S1P3, S1P4, and S1P5) between TSCs and myelinating Schwann cells (MSCs). LPL, which includes lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), is the bioactive lipid that induces a myriad of cellular responses through specific members of G-protein coupled receptors for LPA. It turned out that LPA3 was expressed only in TSCs, whereas S1P1 was expressed in TSCs and skeletal muscle, but not in MSCs. Other types of LPL receptor genes, including LPA1, S1P2, S1P3, S1P4, were expressed in both types of Schwann cells. None of the LPL receptor gene family showed MSCs-specific expression.

  15. Motoneuron development influences dorsal root ganglia survival and Schwann cell development in a vertebrate model of spinal muscular atrophy.

    Science.gov (United States)

    Hao, Le Thi; Duy, Phan Q; Jontes, James D; Beattie, Christine E

    2015-01-15

    Low levels of the survival motor neuron protein (SMN) cause the disease spinal muscular atrophy. A primary characteristic of this disease is motoneuron dysfunction and paralysis. Understanding why motoneurons are affected by low levels of SMN will lend insight into this disease and to motoneuron biology in general. Motoneurons in zebrafish smn mutants develop abnormally; however, it is unclear where Smn is needed for motoneuron development since it is a ubiquitously expressed protein. We have addressed this issue by expressing human SMN in motoneurons in zebrafish maternal-zygotic (mz) smn mutants. First, we demonstrate that SMN is present in axons, but only during the period of robust motor axon outgrowth. We also conclusively demonstrate that SMN acts cell autonomously in motoneurons for proper motoneuron development. This includes the formation of both axonal and dendritic branches. Analysis of the peripheral nervous system revealed that Schwann cells and dorsal root ganglia (DRG) neurons developed abnormally in mz-smn mutants. Schwann cells did not wrap axons tightly and had expanded nodes of Ranvier. The majority of DRG neurons had abnormally short peripheral axons and later many of them failed to divide and died. Expressing SMN just in motoneurons rescued both of these cell types showing that their failure to develop was secondary to the developmental defects in motoneurons. Driving SMN just in motoneurons did not increase survival of the animal, suggesting that SMN is needed for motoneuron development and motor circuitry, but that SMN in other cells types factors into survival. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  16. Concentrated growth factor increases Schwann cell proliferation and neurotrophic factor secretion and promotes functional nerve recovery in vivo.

    Science.gov (United States)

    Qin, Jie; Wang, Lin; Sun, Yue; Sun, Xiaolin; Wen, Chaoju; Shahmoradi, Mahdi; Zhou, Yanmin

    2016-02-01

    Concentrated growth factor (CGF) is a newly generated complex that comprises a fibrin matrix incorporating growth factors and plasmatic and leukocyte cytokines. It has been widely used in bone regenerative medicine. However, the effect of CGF on peripheral nerve regeneration had not been previously investigated. The aim of the present study was to evaluate the possibility of using CGF for nerve regeneration by i) investigating the effect of CGF on the proliferation of Schwann cells (SCs) and secretion of neurotrophic factors nerve growth factor (NGF) and glial cell line‑derived neurotrophic factor (GDNF) in vitro; and ii) analyzing the effect of CGF on functional nerve recovery after nerve injury in vivo. CGF was prepared from venous blood taken from rats, and using scanning electron microscopy (SEM) we noted that it featured a fiber‑like appearance with pore size ranging from 0.1 to 1.0 µm. The soluble component of CGF was used to produce conditioned media with which to treat the Schwann cell line. A cell counting kit-8 assay and cell cycle analysis were both used to study the proliferative effect of CGF on SCs. Reverse transcription-quantitative PCR and western blot analysis demonstrated that there was an increase in the mRNA and protein expression of NGF and GDNF, both of which are markers of SC neurotrophic secretion. A model of sciatic nerve crush injury was established for the in vivo experiment, and CGF was found to increase the sciatic functional index (indicative of nerve function). We noted that CGF increased SC proliferation and secretion of neurotrophic factors in vitro, and promoted functional recovery after peripheral nerve injuries in vivo. These results suggest that CGF is a promising candidate biomaterial for peripheral nerve regeneration, and may potentially be utilized to repair nerve injuries.

  17. Salidroside attenuates colistin-induced neurotoxicity in RSC96 Schwann cells through PI3K/Akt pathway.

    Science.gov (United States)

    Lu, Ziyin; Jiang, Guozheng; Chen, Ying; Wang, Jian; Muhammad, Ishfaq; Zhang, Ling; Wang, Rui; Liu, Fangping; Li, Rui; Qian, Feng; Li, Jichang

    2017-06-01

    Neurotoxicity is a key dose-limiting factor for colistin therapy. This study aimed to investigate the protective effect of Salidroside on colistin-induced neurotoxicity in RSC96 Schwann cells and the underlying mechanisms. After Salidroside (12.5, 25, 50 μg/mL) treatment for 2 h, the cells were cultured with 250 μg/mL colistin for 24 h. In order to investigate the role of phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway, the cells were pre-treated with LY294002 (12.5 μmol/L, a specific inhibitor of PI3K phosphorylation) for 1 h before Salidroside (50 μg/mL) treatment, then were co-cultured with colistin (250 μg/mL) for 24 h. The results showed that colistin treatment could induce apoptotic cell death which was associated with oxidative stress injury. Salidroside could reduce colistin-induced neurotoxicity, decrease the effect of colistin on the reduced expression levels of p-Akt and Bcl-2, and increased the expresion of Bax, release of Cyt c, and activation of caspase-3. However, the protective effect of Salidroside against colistin-induced apoptosis was partly abolished by LY294002. These findings suggest that Salidroside could attenuate colistin-induced neurotoxicity in RSC96 Schwann cells via the PI3K/Akt pathway. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. No shortcuts to pig embryonic stem cells.

    Science.gov (United States)

    Brevini, T A L; Pennarossa, G; Gandolfi, F

    2010-09-01

    The establishment of embryonic stem cell (ESC) lines in domestic species could have great impact in the agricultural as well as in the biomedical field. In particular, derivation of pig ESC would find important applications aimed at improving health and production traits of this species through genetic engineering. Similarly, the immunological, morphological, physiological, and functional similarities to the human make the pig a very effective and suitable animal model for biomedical studies and pre-clinical trials. While proven blastocyst-derived mouse and human ESC lines have been established, no validated porcine ESC (pESC) lines are available. In the present manuscript we briefly discuss some of the factors that make the establishment of ESC lines in the pig, and in animal species other than mouse and human, a very slow process. The paucity of information related to morphology, pluripotency markers, differentiation capability hampers a thorough evaluation of the validity of putative lines. These difficulties are further increased by the lack of reliable antibodies, reagents, and in vitro culture systems that could ensure reliable results in the pig and allow for the screening and long-term maintenance of pESC. Data from the literature suggest that similar regulatory pathways are likely to exist among different species. Coupling of these pathways with their distinct expression patterns, the relative concentrations of pluripotency-related molecules, and timing of embryo development, along with supportive micro-environmental conditions, would appear to vary in a species-specific manner. We feel that the understanding of these subtle but meaningful diversities may provide beneficial information about the isolation of genuine porcine embryonic stem cells. Copyright 2010 Elsevier Inc. All rights reserved.

  19. Transfer of Vesicles From Schwann Cells to Axons: a Novel Mechanism of Communication in the Peripheral Nervous System

    Science.gov (United States)

    Lopez-Verrilli, M. Alejandra; Court, Felipe A.

    2012-01-01

    Schwann cells (SCs) are the glial component of the peripheral nervous system, with essential roles during development and maintenance of axons, as well as during regenerative processes after nerve injury. SCs increase conduction velocities by myelinating axons, regulate synaptic activity at presynaptic nerve terminals and are a source of trophic factors to neurons. Thus, development and maintenance of peripheral nerves are crucially dependent on local signaling between SCs and axons. In addition to the classic mechanisms of intercellular signaling, the possibility of communication through secreted vesicles has been poorly explored to date. Interesting recent findings suggest the occurrence of lateral transfer mediated by vesicles from glial cells to axons that could have important roles in axonal growth and axonal regeneration. Here, we review the role of vesicular transfer from SCs to axons and propose the advantages of this means in supporting neuronal and axonal maintenance and regeneration after nerve damage. PMID:22707941

  20. Transfer of vesicles from Schwann cell to axon: a novel mechanism of communication in the peripheral nervous system

    Directory of Open Access Journals (Sweden)

    María Alejandra eLopez-Verrilli

    2012-06-01

    Full Text Available Schwann cells (SCs are the glial component of the peripheral nervous system, with essential roles during development and maintenance of axons, as well as during regenerative processes after nerve injury. SCs increase conduction velocities by myelinating axons, regulate synaptic activity at presynaptic nerve terminals and are a source of trophic factors to neurons. Thus, development and maintenance of peripheral nerves are crucially dependent on local signalling between SCs and axons. In addition to the classic mechanisms of intercellular signalling, the possibility of communication through secreted vesicles has been poorly explored to date. Interesting recent findings suggest the occurrence of lateral transfer mediated by vesicles from glial cells to axons that could have important roles in axonal growth and axonal regeneration. Here, we review the role of vesicular transfer from SCs to axons and propose the benefits of this means in supporting neuronal and axonal maintenance and regeneration after nerve damage.

  1. Graphene for enhanced embryonic stem cell photo-transfection efficiency

    CSIR Research Space (South Africa)

    Mthunzi, P

    2013-04-01

    Full Text Available Due to their pluripotency properties, embryonic stem (ES) cells possess great potential in regenerative therapy. Since reported a promising tissue engineering scaffold material, here, graphene is demonstrated to significantly improve the ES cell...

  2. A Focused Microarray for Screening Rat Embryonic Stem Cell Lines

    OpenAIRE

    Hong, James; He, Hong; Bui, Phuoc; Ryba-White, Ben; Rumi, Mohammad A.K.; Soares, Michael J.; Dutta, Debasree; Paul, Soumen; Kawamata, Masaki; Ochiya, Takahiro; Ying, Qi-Long; Rajanahalli, Pavan; Mark L. Weiss

    2012-01-01

    Here, we describe a focused microarray for screening rat embryonic stem cells (ESCs) and provide validation data that this array can distinguish undifferentiated rat ESCs from rat trophoblast stem (TS) cells, rat extraembryonic endoderm cells, mouse embryonic fibroblast feeder cells, and differentiated rat ESCs. Using this tool, genuine rat ESC lines, which have been expanded in a conventional rat ESC medium containing two inhibitors (2i), for example, glycogen synthase kinase 3 (GSK3) and mi...

  3. Clinical grade cultivation of human Schwann cell, by the using of human autologous serum instead of fetal bovine serum and without growth factors.

    Science.gov (United States)

    Aghayan, Hamid-Reza; Arjmand, Babak; Norouzi-Javidan, Abbas; Saberi, Hooshang; Soleimani, Masoud; Tavakoli, Seyed Amir-Hossein; Khodadadi, Abbas; Tirgar, Niloufar; Mohammadi-Jahani, Fereshteh

    2012-06-01

    Clinical grade cultivation of human schwann cell by the utilization of human autologous serum instead of fetal bovine serum, and also avoiding any growth factors, can increase safety level of this procedure in cases of clinical cell transplantation. The aim of this study was demonstration of the feasibility of clinical grade schwann cell cultivation. In this experimental study after obtaining consent from close relatives we harvested 10 sural nerves from brain death donors and then cultured in 10 seperated culture media plus autologous serum. We also prepared autologous serum from donor's whole blood. Then cultured cells were evaluated by S100 antibody staining for both morphology and purity. Cell purity range was from 97% to 99% (mean=98.11 ± 0.782%). Mean of the cell count was 14,055.56 ± 2,480.479 per micro liter. There was not significant correlation between cell purity and either the culture period or the age of donors (P>0.05). The spearman correlation coefficient for the cell purity with the period or the age of donors was 0.21 and 0.09, respectively. We demonstrated the feasibility of clinical grade schwann cell cultivation by the using of human autologous serum instead of fetal bovine serum and also without the using of growth factors. We also recommended all cell preparation facilities to adhere to the GMP and other similar quality disciplines especially in the preparation of clinically-used cell products.

  4. Methylcobalamin promotes the differentiation of Schwann cells and remyelination in lysophosphatidylcholine-induced demyelination of the rat sciatic nerve

    Directory of Open Access Journals (Sweden)

    Shunsuke eNishimoto

    2015-08-01

    Full Text Available Schwann cells (SCs are constituents of the peripheral nervous system. The differentiation of SCs in injured peripheral nerves is critical for regeneration after injury. Methylcobalamin (MeCbl is a vitamin B12 analog that is necessary for the maintenance of the peripheral nervous system. In this study, we estimated the effect of MeCbl on SCs. We showed that MeCbl downregulated the activity of Erk1/2 and promoted the expression of the myelin basic protein in SCs. In a dorsal root ganglion neuron–SC coculture system, myelination was promoted by MeCbl. In a focal demyelination rat model, MeCbl promoted remyelination and motor and sensory functional regeneration. MeCbl promoted the in vitro differentiation of SCs and in vivo myelination in a rat demyelination model and may be a novel therapy for several types of nervous disorders.

  5. Rat Sciatic Nerve Reconstruction Across a 30 mm Defect Bridged by an Oriented Porous PHBV Tube With Schwann Cell as Artificial Nerve Graft

    OpenAIRE

    Karimi, Mina; Biazar, Esmaeil; Keshel, Saeed Heidari; Ronaghi, Abdolaziz; Doostmohamadpour, Jafar; Janfada, Alireza; Montazeri, Arash

    2014-01-01

    An oriented poly(3-hydroxybutyrate-co-3-hydroxyvalerate) nerve conduit has been used to evaluate its efficiency based on the promotion of peripheral nerve regeneration in rats. The oriented porous micropatterned artificial nerve conduit was designed onto the micropatterned silicon wafers, and then their surfaces were modified with oxygen plasma to increase cell adhesion. The designed conduits were investigated by cell culture analyses with Schwann cells (SCs). The conduits were implanted into...

  6. Embryonic retinal cells and support to mature retinal neurons.

    Science.gov (United States)

    Stanke, Jennifer J; Fischer, Andy J

    2010-04-01

    Purpose. There is a paucity of neuron replacement studies for retinal ganglion cells. Given the complex phenotype of these neurons, replacement of ganglion cells may be impossible. However, transplanted embryonic cells could provide factors that promote the survival of these neurons. The authors sought to determine whether transplanted embryonic retinal cells from various stages of development influence the survival of mature ganglion cells Methods. Acutely dissociated retinal cells, obtained from chick embryos, were transplanted into the vitreous chamber of posthatch chicken eyes after the ganglion cells were selectively damaged. Eight days after transplantation, numbers of ganglion cells were determined Results. Embryonic retinal cells from embryonic day (E)7, E10, and E11 promoted the survival of ganglion cells, whereas cells from earlier or later stages of development or from other tissue sources did not. The environment provided by the posthatch eye did not support the proliferation of the embryo-derived cells, unlike the environment provided by culture conditions. Furthermore, cells that migrated into the retina failed to express neuronal or glial markers; those that remained in the vitreous formed aggregates of neuronal and glial cells Conclusions. The environment provided within the mature retina does not support the differentiation and proliferation of retinal progenitors. Furthermore, embryo-derived cells likely produce secreted factors that promote the survival of damaged ganglion cells. Therefore, embryonic retinal cells could be applied as a cell-based survival therapy to treat neurodegenerative diseases of the retina.

  7. Cathepsin E generates a sumoylated intracellular fragment of the cell adhesion molecule L1 to promote neuronal and Schwann cell migration as well as myelination.

    Science.gov (United States)

    Lutz, David; Wolters-Eisfeld, Gerrit; Schachner, Melitta; Kleene, Ralf

    2014-03-01

    The cell adhesion molecule L1 regulates cellular responses in the developing and adult nervous system. Here, we show that stimulation of cultured mouse cerebellar neurons by a function-triggering L1 antibody leads to cathepsin E-mediated generation of a sumoylated 30 kDa L1 fragment (L1-30) and to import of L1-30 into the nucleus. Mutation of the sumoylation site at K1172 or the cathepsin E cleavage site at E1167 abolishes generation of L1-30, while mutation of the nuclear localization signal at K1147 prevents nuclear import of L1-30. Moreover, the aspartyl protease inhibitor pepstatin impairs the generation of L1-30 and inhibits L1-induced migration of cerebellar neurons and Schwann cells as well as L1-dependent in vitro myelination on axons of dorsal root ganglion neurons by Schwann cells. L1-stimulated migration of HEK293 cells expressing L1 with mutated cathepsin E cleavage site is diminished in comparison to migration of cells expressing non-mutated L1. In addition, L1-stimulated migration of HEK293 cells expressing non-mutated L1 is also abolished upon knock-down of cathepsin E expression and enhanced by over-expression of cathepsin E. The findings of the present study indicate that generation and nuclear import of L1-30 regulate neuronal and Schwann cell migration as well as myelination. Cell adhesion molecule L1 regulates cellular responses in the developing and adult nervous system. L1 stimulation triggers sumoylation and cleavage of L1, thus generating the L1-70 fragment (1) which is cleaved by cathepsin E (2) yielding the L1-30 fragment that is imported to the nucleus (3), may bind to DNA and/or nuclear proteins (4), to regulate diverse cellular functions. © 2013 International Society for Neurochemistry.

  8. p75(NTR) expression and nuclear localization of p75(NTR) intracellular domain in spiral ganglion Schwann cells following deafness correlate with cell proliferation.

    Science.gov (United States)

    Provenzano, Matthew J; Minner, Sarah A; Zander, Kaitlin; Clark, J Jason; Kane, Catherine J; Green, Steven H; Hansen, Marlan R

    2011-08-01

    Spiral ganglion Schwann cells (SGSCs) myelinate spiral ganglion neurons (SGNs) and represent a potential source of neurotrophic support for SGNs. Deafening due to loss of hair cells results in gradual degeneration and death of SGNs. Successful efforts to maintain or regenerate a functional auditory nerve may depend on a healthy population of SGSCs, yet the responses of SGSCs to neural injury remain largely unknown. Here we investigate the role of p75(NTR) in SGSC responses to gradual denervation. Following deafening, SGSCs in the osseous spiral lamina (OSL) and, subsequently, in Rosenthal's canal (RC) expressed elevated p75(NTR) compared to hearing controls. p75(NTR)-positive cells co-labeled with S100 and RIP antibodies (Schwann cell markers), but not with anti-neurofilament. The pattern of p75(NTR) expression mirrored the pattern of neural degeneration, beginning in the OSL of the cochlea base and later extending into the apex. SGSCs expressed sortilin, a p75(NTR) co-receptor for pro-neurotrophins. Both pro-nerve growth factor (pro-NGF) and pro-brain derived neurotrophic factor (proBDNF) induced apoptosis in cultured SGSCs. Deafened animals exhibited significantly higher levels of SGSC proliferation (as measured by BrdU uptake) compared to hearing animals while total Schwann cell density remained stable, suggesting a tight regulation of SGSC proliferation and cell death. SGSCs undergoing cell division lose p75(NTR) expression from the cell surface and demonstrate nuclear localization of the intracellular domain (ICD), raising the possibility that p75(NTR) cleavage and ICD nuclear localization regulate SGSC proliferation. These results suggest that p75(NTR) contributes to SGSC responses to deafening and neural degeneration. Copyright © 2011 Elsevier Inc. All rights reserved.

  9. The Effect of Electrospun Gelatin Fibers Alignment on Schwann Cell and Axon Behavior and Organization in the Perspective of Artificial Nerve Design

    Directory of Open Access Journals (Sweden)

    Sara Gnavi

    2015-06-01

    Full Text Available Electrospun fibrous substrates mimicking extracellular matrices can be prepared by electrospinning, yielding aligned fibrous matrices as internal fillers to manufacture artificial nerves. Gelatin aligned nano-fibers were prepared by electrospinning after tuning the collector rotation speed. The effect of alignment on cell adhesion and proliferation was tested in vitro using primary cultures, the Schwann cell line, RT4-D6P2T, and the sensory neuron-like cell line, 50B11. Cell adhesion and proliferation were assessed by quantifying at several time-points. Aligned nano-fibers reduced adhesion and proliferation rate compared with random fibers. Schwann cell morphology and organization were investigated by immunostaining of the cytoskeleton. Cells were elongated with their longitudinal body parallel to the aligned fibers. B5011 neuron-like cells were aligned and had parallel axon growth when cultured on the aligned gelatin fibers. The data show that the alignment of electrospun gelatin fibers can modulate Schwann cells and axon organization in vitro, suggesting that this substrate shows promise as an internal filler for the design of artificial nerves for peripheral nerve reconstruction.

  10. The Effect of Electrospun Gelatin Fibers Alignment on Schwann Cell and Axon Behavior and Organization in the Perspective of Artificial Nerve Design.

    Science.gov (United States)

    Gnavi, Sara; Fornasari, Benedetta Elena; Tonda-Turo, Chiara; Laurano, Rossella; Zanetti, Marco; Ciardelli, Gianluca; Geuna, Stefano

    2015-06-08

    Electrospun fibrous substrates mimicking extracellular matrices can be prepared by electrospinning, yielding aligned fibrous matrices as internal fillers to manufacture artificial nerves. Gelatin aligned nano-fibers were prepared by electrospinning after tuning the collector rotation speed. The effect of alignment on cell adhesion and proliferation was tested in vitro using primary cultures, the Schwann cell line, RT4-D6P2T, and the sensory neuron-like cell line, 50B11. Cell adhesion and proliferation were assessed by quantifying at several time-points. Aligned nano-fibers reduced adhesion and proliferation rate compared with random fibers. Schwann cell morphology and organization were investigated by immunostaining of the cytoskeleton. Cells were elongated with their longitudinal body parallel to the aligned fibers. B5011 neuron-like cells were aligned and had parallel axon growth when cultured on the aligned gelatin fibers. The data show that the alignment of electrospun gelatin fibers can modulate Schwann cells and axon organization in vitro, suggesting that this substrate shows promise as an internal filler for the design of artificial nerves for peripheral nerve reconstruction.

  11. Cultures of Schwann-like cells differentiated from adipose-derived stem cells on PDMS/MWNT sheets as a scaffold for peripheral nerve regeneration.

    Science.gov (United States)

    Han, In Ho; Sun, Fangfang; Choi, Yoon Ji; Zou, Fengming; Nam, Kyoung Hyup; Cho, Won Ho; Choi, Byung Kwan; Song, Geun Sung; Koh, Kwangnak; Lee, Jaebeom

    2015-11-01

    Carbon nanotubes (CNTs) are promising candidates as novel scaffolds for peripheral nerve regeneration. Schwann cells (SCs) are attractive therapeutic targets due to their pivotal role in peripheral nerve regeneration, but primary SCs have limitations for clinical application. However, adipose-derived stem cells (ASCs) may differentiate into Schwann-like cells. The present study assesses the potential applicability of multiwall CNTs (MWNTs) composited with polydimethylsiloxane (PDMS), which were then seeded with differentiated adipose-derived stem cells (dASCs) to promote neuronal differentiation and growth. Aqueous MWNT dispersion was filtered, and the PDMS/MWNT sheets were prepared using a simple printing-transfer method. Characterization of PDMS/MWNT sheets indicated their unique physical properties, such as superior mechanical strength and electroconductivity, compared with bare PDMS sheets. ASCs were differentiated into Schwann-like cells using a mixture of glial growth factors. Dorsal root ganglion (DRG) neurons were co-cultured with SCs and dASCs on PDMS/MWNTs sheets or noncoated dishes. An alamar blue proliferation assay of dASC and SCs showed significantly more dASC and SCs cultured on PDMS/MWNT sheets at 48 h and 72 h than when cultured on noncoated dishes (p proliferation of DRG neurons and the longest neurite outgrowth length per neuron were significantly greater than when DRG were cultured on PDMS/MWNT sheets alone or on noncoated dishes seeded with SCs or dASCs (p cells differentiated from ASCs. Seeding the dASCs on PDMS/MWNT sheets may produce synergistic effects in peripheral nerve regeneration, similarly to SCs. © 2015 Wiley Periodicals, Inc.

  12. Dynamic Proteomic Profiling of Extra-Embryonic Endoderm Differentiation in Mouse Embryonic Stem Cells.

    Science.gov (United States)

    Mulvey, Claire M; Schröter, Christian; Gatto, Laurent; Dikicioglu, Duygu; Fidaner, Isik Baris; Christoforou, Andy; Deery, Michael J; Cho, Lily T Y; Niakan, Kathy K; Martinez-Arias, Alfonso; Lilley, Kathryn S

    2015-09-01

    During mammalian preimplantation development, the cells of the blastocyst's inner cell mass differentiate into the epiblast and primitive endoderm lineages, which give rise to the fetus and extra-embryonic tissues, respectively. Extra-embryonic endoderm (XEN) differentiation can be modeled in vitro by induced expression of GATA transcription factors in mouse embryonic stem cells. Here, we use this GATA-inducible system to quantitatively monitor the dynamics of global proteomic changes during the early stages of this differentiation event and also investigate the fully differentiated phenotype, as represented by embryo-derived XEN cells. Using mass spectrometry-based quantitative proteomic profiling with multivariate data analysis tools, we reproducibly quantified 2,336 proteins across three biological replicates and have identified clusters of proteins characterized by distinct, dynamic temporal abundance profiles. We first used this approach to highlight novel marker candidates of the pluripotent state and XEN differentiation. Through functional annotation enrichment analysis, we have shown that the downregulation of chromatin-modifying enzymes, the reorganization of membrane trafficking machinery, and the breakdown of cell-cell adhesion are successive steps of the extra-embryonic differentiation process. Thus, applying a range of sophisticated clustering approaches to a time-resolved proteomic dataset has allowed the elucidation of complex biological processes which characterize stem cell differentiation and could establish a general paradigm for the investigation of these processes. © 2015 AlphaMed Press.

  13. Differential astroglial responses in the spinal cord of rats submitted to a sciatic nerve double crush treated with local injection of cultured Schwann cell suspension or lesioned spinal cord extract: implications on cell therapy for nerve repair Respostas astrocitárias na medula espinal do rato submetido ao esmagamento duplo do nervo ciático e tratado com injeção local de suspensão de células de Schwann cultivadas ou de extrato de medula espinal lesada: implicações na terapia celular para o reparo do nervo

    National Research Council Canada - National Science Library

    João Gabriel Martins Dallo; Bernardo Vergara Reichert; José Benedito Ramos Valladão Júnior; Camila Silva; Bianca Aparecida de Luca; Beatriz de Freitas Azevedo Levy; Gerson Chadi

    2007-01-01

    PURPOSE: Reactive astrocytes are implicated in several mechanisms after central or peripheral nervous system lesion, including neuroprotection, neuronal sprouting, neurotransmission and neuropathic pain. Schwann cells (SC...

  14. In vitro differentiation of mouse embryonic stem cells into functional ...

    African Journals Online (AJOL)

    Jane

    2011-08-22

    Aug 22, 2011 ... Studies have shown that embryonic stem (ES) cells can be successfully differentiated into liver cells, which offer the potential unlimited cell source for a variety of end-stage liver disease. In our study, in order to induce mouse ES cells to differentiate into hepatocyte-like cells under chemically defined.

  15. Human Embryonic Stem Cell Research Debates: A Confucian Argument

    National Research Council Canada - National Science Library

    D. F.-C. Tsai

    2005-01-01

    Human embryonic stem cell research can bring about major biomedical breakthroughs and thus contribute enormously to human welfare, yet it raises serious moral problems because it involves using human...

  16. Nucleosome Organization in Human Embryonic Stem Cells.

    Directory of Open Access Journals (Sweden)

    Puya G Yazdi

    Full Text Available The fundamental repeating unit of eukaryotic chromatin is the nucleosome. Besides being involved in packaging DNA, nucleosome organization plays an important role in transcriptional regulation and cellular identity. Currently, there is much debate about the major determinants of the nucleosome architecture of a genome and its significance with little being known about its role in stem cells. To address these questions, we performed ultra-deep sequencing of nucleosomal DNA in two human embryonic stem cell lines and integrated our data with numerous epigenomic maps. Our analyses have revealed that the genome is a determinant of nucleosome organization with transcriptionally inactive regions characterized by a "ground state" of nucleosome profiles driven by underlying DNA sequences. DNA sequence preferences are associated with heterogeneous chromatin organization around transcription start sites. Transcription, histone modifications, and DNA methylation alter this "ground state" by having distinct effects on both nucleosome positioning and occupancy. As the transcriptional rate increases, nucleosomes become better positioned. Exons transcribed and included in the final spliced mRNA have distinct nucleosome profiles in comparison to exons not included at exon-exon junctions. Genes marked by the active modification H3K4m3 are characterized by lower nucleosome occupancy before the transcription start site compared to genes marked by the inactive modification H3K27m3, while bivalent domains, genes associated with both marks, lie exactly in the middle. Combinatorial patterns of epigenetic marks (chromatin states are associated with unique nucleosome profiles. Nucleosome organization varies around transcription factor binding in enhancers versus promoters. DNA methylation is associated with increasing nucleosome occupancy and different types of methylations have distinct location preferences within the nucleosome core particle. Finally, computational

  17. Characterization and Schwann Cell Seeding of up to 15.0 cm Long Spider Silk Nerve Conduits for Reconstruction of Peripheral Nerve Defects

    Directory of Open Access Journals (Sweden)

    Tim Kornfeld

    2016-11-01

    Full Text Available Nerve reconstruction of extended nerve defect injuries still remains challenging with respect to therapeutic options. The gold standard in nerve surgery is the autologous nerve graft. Due to the limitation of adequate donor nerves, surgical alternatives are needed. Nerve grafts made out of either natural or artificial materials represent this alternative. Several biomaterials are being explored and preclinical and clinical applications are ongoing. Unfortunately, nerve conduits with successful enhancement of axonal regeneration for nerve defects measuring over 4.0 cm are sparse and no conduits are available for nerve defects extending to 10.0 cm. In this study, spider silk nerve conduits seeded with Schwann cells were investigated for in vitro regeneration on defects measuring 4.0 cm, 10.0 cm and 15.0 cm in length. Schwann cells (SCs were isolated, cultured and purified. Cell purity was determined by immunofluorescence. Nerve grafts were constructed out of spider silk from Nephila edulis and decellularized ovine vessels. Finally, spider silk implants were seeded with purified Schwann cells. Cell attachment was observed within the first hour. After 7 and 21 days of culture, immunofluorescence for viability and determination of Schwann cell proliferation and migration throughout the conduits was performed. Analyses revealed that SCs maintained viable (>95% throughout the conduits independent of construct length. SC proliferation on the spider silk was determined from day 7 to day 21 with a proliferation index of 49.42% arithmetically averaged over all conduits. This indicates that spider silk nerve conduits represent a favorable environment for SC attachment, proliferation and distribution over a distance of least 15.0 cm in vitro. Thus spider silk nerve implants are a highly adequate biomaterial for nerve reconstruction.

  18. Characterization and Schwann Cell Seeding of up to 15.0 cm Long Spider Silk Nerve Conduits for Reconstruction of Peripheral Nerve Defects

    Science.gov (United States)

    Kornfeld, Tim; Vogt, Peter M.; Bucan, Vesna; Peck, Claas-Tido; Reimers, Kerstin; Radtke, Christine

    2016-01-01

    Nerve reconstruction of extended nerve defect injuries still remains challenging with respect to therapeutic options. The gold standard in nerve surgery is the autologous nerve graft. Due to the limitation of adequate donor nerves, surgical alternatives are needed. Nerve grafts made out of either natural or artificial materials represent this alternative. Several biomaterials are being explored and preclinical and clinical applications are ongoing. Unfortunately, nerve conduits with successful enhancement of axonal regeneration for nerve defects measuring over 4.0 cm are sparse and no conduits are available for nerve defects extending to 10.0 cm. In this study, spider silk nerve conduits seeded with Schwann cells were investigated for in vitro regeneration on defects measuring 4.0 cm, 10.0 cm and 15.0 cm in length. Schwann cells (SCs) were isolated, cultured and purified. Cell purity was determined by immunofluorescence. Nerve grafts were constructed out of spider silk from Nephila edulis and decellularized ovine vessels. Finally, spider silk implants were seeded with purified Schwann cells. Cell attachment was observed within the first hour. After 7 and 21 days of culture, immunofluorescence for viability and determination of Schwann cell proliferation and migration throughout the conduits was performed. Analyses revealed that SCs maintained viable (>95%) throughout the conduits independent of construct length. SC proliferation on the spider silk was determined from day 7 to day 21 with a proliferation index of 49.42% arithmetically averaged over all conduits. This indicates that spider silk nerve conduits represent a favorable environment for SC attachment, proliferation and distribution over a distance of least 15.0 cm in vitro. Thus spider silk nerve implants are a highly adequate biomaterial for nerve reconstruction. PMID:27916868

  19. GDNF Schwann cells in hydrogel scaffolds promote regional axon regeneration, remyelination and functional improvement after spinal cord transection in rats.

    Science.gov (United States)

    Chen, Bingkun K; Madigan, Nicolas N; Hakim, Jeffrey S; Dadsetan, Mahrokh; McMahon, Siobhan S; Yaszemski, Michael J; Windebank, Anthony J

    2017-03-10

    Positively-charged oligo[poly(ethylene glycol)fumarate] (OPF+ ) is a biodegradable hydrogel used for spinal cord injury repair. We compared scaffolds containing primary Schwann cells (SCs) to scaffolds delivering SCs genetically modified to secrete high concentrations of glial cell-derived neurotrophic factor (GDNF). Multichannel OPF+ scaffolds loaded with SCs or GDNF-SCs were implanted into transected rat spinal cords for 4 weeks. GDNF-SCs promoted regeneration of more axons into OPF+ scaffolds (2773.0 ± 396.0) than primary SC OPF+ scaffolds (1666.0 ± 352.2) (p = 0.0491). This increase was most significant in central and ventral-midline channels of the scaffold. Axonal remyelination was quantitated by stereologic analysis. Increased myelination of regenerating axons was observed in the GDNF-SC group. Myelinating cell and axon complexes were formed by host SCs and not by implanted cells or host oligodendrocytes. Fast Blue retrograde tracing studies determined the rostral-caudal directionality of axonal growth. The number of neurons that projected axons rostrally through the GDNF-SC scaffolds was higher (7929 ± 1670) than in animals with SC OPF+ scaffolds (1069 ± 241.5) (p OPF+ scaffolds partially recovered locomotor function at weeks 3 and 4 following surgery. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  20. Regulated viral BDNF delivery in combination with Schwann cells promotes axonal regeneration through capillary alginate hydrogels after spinal cord injury.

    Science.gov (United States)

    Liu, Shengwen; Sandner, Beatrice; Schackel, Thomas; Nicholson, LaShae; Chtarto, Abdelwahed; Tenenbaum, Liliane; Puttagunta, Radhika; Müller, Rainer; Weidner, Norbert; Blesch, Armin

    2017-09-15

    Grafting of cell-seeded alginate capillary hydrogels into a spinal cord lesion site provides an axonal bridge while physically directing regenerating axonal growth in a linear pattern. However, without an additional growth stimulus, bridging axons fail to extend into the distal host spinal cord. Here we examined whether a combinatory strategy would support regeneration of descending axons across a cervical (C5) lateral hemisection lesion in the rat spinal cord. Following spinal cord transections, Schwann cell (SC)-seeded alginate hydrogels were grafted to the lesion site and AAV5 expressing brain-derived neurotrophic factor (BDNF) under control of a tetracycline-regulated promoter was injected caudally. In addition, we examined whether SC injection into the caudal spinal parenchyma would further enhance regeneration of descending axons to re-enter the host spinal cord. Our data show that both serotonergic and descending axons traced by biotinylated dextran amine (BDA) extend throughout the scaffolds. The number of regenerating axons is significantly increased when caudal BDNF expression is activated and transient BDNF delivery is able to sustain axons after gene expression is switched off. Descending axons are confined to the caudal graft/host interface even with continuous BDNF expression for 8weeks. Only with a caudal injection of SCs, a pathway facilitating axonal regeneration through the host/graft interface is generated allowing axons to successfully re-enter the caudal spinal cord. Recovery from spinal cord injury is poor due to the limited regeneration observed in the adult mammalian central nervous system. Biomaterials, cell transplantation and growth factors that can guide axons across a lesion site, provide a cellular substrate, stimulate axon growth and have shown some promise in increasing the growth distance of regenerating axons. In the present study, we combined an alginate biomaterial with linear channels with transplantation of Schwann cells within

  1. Tumor-suppression functions of merlin are independent of its role as an organizer of the actin cytoskeleton in Schwann cells.

    Science.gov (United States)

    Lallemand, Dominique; Saint-Amaux, Aurelie Lampin; Giovannini, Marco

    2009-11-15

    Merlin is the product of the Nf2 tumor-suppressor gene, and inactivation of Nf2 leads to the development of neural tumors such as schwannomas and meningiomas in humans and mice. Merlin is a member of the ERM (ezrin, radixin and moesin) family of proteins that function as organizers of the actin cytoskeleton. Merlin structure is thought to be similar to that of the ERM proteins, and is held in a closed clamp conformation via intramolecular interactions of its N-terminal FERM (four-point-one, ERM) domain with an alpha-helical C-terminal domain. Like ERMs, merlin can remodel actin-rich cortical structures, yet merlin uniquely inhibits the proliferation of many different cell types. Here, we report that the F2 subdomain of the FERM domain and a domain close to the C-terminus that is defined by residues 532-579 are essential for merlin-mediated inhibition of primary Schwann cell proliferation. Furthermore, we demonstrate that the F1 subdomain of the merlin FERM domain is required for actin colocalization, proper regulation of merlin C-terminal phosphorylation and for remodeling the cytoskeleton, yet is not required for the inhibition of Schwann cell proliferation. Thus, tumor suppression by merlin is independent of its role as an organizer of the actin cytoskeleton in Schwann cells.

  2. A novel marker for terminal Schwann cells, homocysteine-responsive ER-resident protein, as isolated by a single cell PCR-differential display.

    Science.gov (United States)

    Oda, Ryo; Yaoi, Takeshi; Okajima, Seiichiro; Kobashi, Hiroaki; Kubo, Toshikazu; Fushiki, Shinji

    2003-09-05

    Terminal Schwann cells (TSCs) that cover motor neuron terminals are known to play important roles in maintaining neuromuscular junctions, as well as in the repair process after nerve injury. However, molecular characteristics of TSCs remain unknown, because of the difficulties in analyzing them due to their paucity. We have established a method of selectively and efficiently collecting TSCs so that cDNA analysis can be done properly. The expression of 1-2% of whole mRNAs was compared between myelinating Schwann cells (MSCs) and TSCs, and it turned out that approximately one-third of the bands could be categorized as cell-type-specific bands. TSCs thus constitute a distinct entity from the viewpoint of gene expression. As one of the cDNA clones belonging to TSC-specific bands was identified homocysteine-responsive ER-resident protein (Herp), and in situ hybridization confirmed that Herp mRNA is expressed in TSCs on motor nerve terminals but not in MSCs, both in developing and adult rats. In conclusion, we have been able to identify Herp as a novel molecular marker for TSCs.

  3. The Use of Fiber-Reinforced Scaffolds Cocultured with Schwann Cells and Vascular Endothelial Cells to Repair Rabbit Sciatic Nerve Defect with Vascularization

    Directory of Open Access Journals (Sweden)

    Hongyang Gao

    2013-01-01

    Full Text Available To explore the feasibility of biodegradable fiber-reinforced 3D scaffolds with satisfactory mechanical properties for the repair of long-distance sciatic nerve defect in rabbits and effects of vascularized graft in early stage on the recovery of neurological function, Schwann cells and vascular endothelial cells were cocultured in the fiber-reinforced 3D scaffolds. Experiment group which used prevascularized nerve complex for the repair of sciatic nerve defect and control group which only cultured with Schwann cells were set. The animals in both groups underwent electromyography to show the status of the neurological function recovery at 4, 8, and 16 weeks after the surgery. Sciatic nerve regeneration and myelination were observed under the light microscope and electron microscope. Myelin sheath thickness, axonal diameter, and number of myelinated nerve fiber were quantitatively analyzed using image analysis system. The recovery of foot ulcer, the velocity of nerve conduction, the number of regenerating nerve fiber, and the recovery of ultrastructure were increased in the experimental group than those in the control group. Prevascularized tissue engineered fiber-reinforced 3D scaffolds for the repair of sciatic nerve defects in rabbits can effectively promote the recovery of neurological function.

  4. p53 regulation and activity in mouse embryonic stem cells

    OpenAIRE

    Solozobova, Valeriya

    2010-01-01

    P53 is a tumour development p53. The aim of this work was to study the regulation of p53 in embryonic stem cells and its activation in response to DNA damage. p53 was found that p53 becomes transcriptionally active in ES cells after DNA damage. Embryonic stem cells contain a relatively high amount of p53 protein and p53 RNA. After differentiation p53 level is rapidly downregulated. The high abundance of p53 in undifferentiated ES cells is a result of enhanced translation.

  5. Twenty years of embryonic stem cell research in farm animals

    Science.gov (United States)

    Notable distinctions between an embryonic stem cell (ESC) and somatic cell are that the ESC can maintain an undifferentiated state indefinitely, self renew, and is pluripotent, meaning that the ESC can potentially generate cells representing all the three primordial germ layers and contribute to the...

  6. Directional differentiation of chicken embryonic stem cells into ...

    African Journals Online (AJOL)

    Chicken embryonic stem (ES) cells are useful for producing transgenic chickens and preserving genetic material in avian species. In this study, the differentiation potential of chicken ES cells was investigated in vitro. Chicken ES cells were differentiated into osteoblasts cultured for 15 to 21 days in the induction media ...

  7. Salidroside promotes peripheral nerve regeneration based on tissue engineering strategy using Schwann cells and PLGA: in vitro and in vivo

    Science.gov (United States)

    Liu, Hui; Lv, Peizhen; Zhu, Yongjia; Wu, Huayu; Zhang, Kun; Xu, Fuben; Zheng, Li; Zhao, Jinmin

    2017-01-01

    Salidriside (SDS), a phenylpropanoid glycoside derived from Rhodiola rosea L, has been shown to be neuroprotective in many studies, which may be promising in nerve recovery. In this study, the neuroprotective effects of SDS on engineered nerve constructed by Schwann cells (SCs) and Poly (lactic-co-glycolic acid) (PLGA) were studied in vitro. We further investigated the effect of combinational therapy of SDS and PLGA/SCs based tissue engineering on peripheral nerve regeneration based on the rat model of nerve injury by sciatic transection. The results showed that SDS dramatically enhanced the proliferation and function of SCs. The underlying mechanism may be that SDS affects SCs growth through the modulation of neurotrophic factors (BDNF, GDNF and CNTF). 12 weeks after implantation with a 12 mm gap of sciatic nerve injury, SDS-PLGA/SCs achieved satisfying outcomes of nerve regeneration, as evidenced by morphological and functional improvements upon therapy by SDS, PLGA/SCs or direct suture group assessed by sciatic function index, nerve conduction assay, HE staining and immunohistochemical analysis. Our results demonstrated the significant role of introducing SDS into neural tissue engineering to promote nerve regeneration.

  8. Neuregulin1 displayed on motor axons regulates terminal Schwann cell-mediated synapse elimination at developing neuromuscular junctions

    Science.gov (United States)

    Li, Yue; Mikesh, Michelle; Smith, Ian; Nave, Klaus-Armin; Schwab, Markus H.; Thompson, Wesley J.

    2016-01-01

    Synaptic connections in the nervous system are rearranged during development and in adulthood as a feature of growth, plasticity, aging, and disease. Glia are implicated as active participants in these changes. Here we investigated a signal that controls the participation of peripheral glia, the terminal Schwann cells (SCs), at the neuromuscular junction (NMJ) in mice. Transgenic manipulation of the levels of membrane-tethered neuregulin1 (NRG1-III), a potent activator of SCs normally presented on motor axons, alters the rate of loss of motor inputs at NMJs during developmental synapse elimination. In addition, NMJs of adult transgenic mice that expressed excess axonal NRG1-III exhibited continued remodeling, in contrast to the more stable morphologies of controls. In fact, synaptic SCs of these adult mice with NRG1-III overexpression exhibited behaviors evident in wild type neonates during synapse elimination, including an affinity for the postsynaptic myofiber surface and phagocytosis of nerve terminals. Given that levels of NRG1-III expression normally peak during the period of synapse elimination, our findings identify axon-tethered NRG1 as a molecular determinant for SC-driven neuromuscular synaptic plasticity. PMID:26755586

  9. Cdc42 regulates Schwann cell radial sorting and myelin sheath folding through NF2/merlin-dependent and independent signaling.

    Science.gov (United States)

    Guo, Li; Moon, Chandra; Zheng, Yi; Ratner, Nancy

    2013-11-01

    The Rho family GTPase Cdc42 has been implicated in developmental Schwann cell (SC) proliferation, providing sufficient SCs for radial sorting of axons preceding SC differentiation in the peripheral nervous system. We generated Cdc42 conditional knockout (Cdc42-CKO) mice and confirmed aberrant axon sorting in Cdc42-CKO nerves. In adult Cdc42-CKO nerves, blood vessels were enlarged, and mature Remak bundles containing small axons were absent. Abnormal infoldings and outfoldings of myelin sheaths developed in Cdc42-CKO nerves, mimicking pathological features of Charcot-Marie-Tooth (CMT) disease. The NF2/merlin tumor suppressor has been implicated up- and down-stream of Cdc42. In Cdc42-CKO;NF2-del double mutant mice, radial sorting defects seen in Cdc42-CKO nerves were rescued, while changes in myelin sheaths in Cdc42-CKO nerves were not. Phosphorylation of Focal adhesion kinase (FAK) and P-GSK3β, as well as expression of β-catenin were decreased in Cdc42-CKO nerves, and these changes were rescued by NF2/merlin mutation in Cdc42-CKO;NF2-del double mutant mice. Thus, Cdc42 regulates SC radial sorting in vivo through NF2/merlin dependent signaling pathways, while Cdc42 modulation of myelin sheath folding is NF2/merlin independent. Copyright © 2013 Wiley Periodicals, Inc.

  10. Neurotropin® Accelerates the Differentiation of Schwann Cells and Remyelination in a Rat Lysophosphatidylcholine-Induced Demyelination Model

    Directory of Open Access Journals (Sweden)

    Hozo Matsuoka

    2018-02-01

    Full Text Available Neurotropin® (NTP, a non-protein extract of inflamed rabbit skin inoculated with vaccinia virus, is clinically used for the treatment of neuropathic pain in Japan and China, although its effect on peripheral nerve regeneration remains to be elucidated. The purpose of this study was to investigate the effects of NTP on Schwann cells (SCs in vitro and in vivo, which play an important role in peripheral nerve regeneration. In SCs, NTP upregulated protein kinase B (AKT activity and Krox20 and downregulated extracellular signal-regulated kinase1/2 activity under both growth and differentiation conditions, enhanced the expression of myelin basic protein and protein zero under the differentiation condition. In a co-culture of dorsal root ganglion neurons and SCs, NTP accelerated myelination of SCs. To further investigate the influence of NTP on SCs in vivo, lysophosphatidylcholine was injected into the rat sciatic nerve, leading to the focal demyelination. After demyelination, NTP was administered systemically with an osmotic pump for one week. NTP improved the ratio of myelinated axons and motor, sensory, and electrophysiological function. These findings reveal novel effects of NTP on SCs differentiation in vitro and in vivo, and indicate NTP as a promising treatment option for peripheral nerve injuries and demyelinating diseases.

  11. CXCL12α/SDF-1 from perisynaptic Schwann cells promotes regeneration of injured motor axon terminals.

    Science.gov (United States)

    Negro, Samuele; Lessi, Francesca; Duregotti, Elisa; Aretini, Paolo; La Ferla, Marco; Franceschi, Sara; Menicagli, Michele; Bergamin, Elisanna; Radice, Egle; Thelen, Marcus; Megighian, Aram; Pirazzini, Marco; Mazzanti, Chiara M; Rigoni, Michela; Montecucco, Cesare

    2017-08-01

    The neuromuscular junction has retained through evolution the capacity to regenerate after damage, but little is known on the inter-cellular signals involved in its functional recovery from trauma, autoimmune attacks, or neurotoxins. We report here that CXCL12α, also abbreviated as stromal-derived factor-1 (SDF-1), is produced specifically by perisynaptic Schwann cells following motor axon terminal degeneration induced by α-latrotoxin. CXCL12α acts via binding to the neuronal CXCR4 receptor. A CXCL12α-neutralizing antibody or a specific CXCR4 inhibitor strongly delays recovery from motor neuron degeneration in vivo Recombinant CXCL12α in vivo accelerates neurotransmission rescue upon damage and very effectively stimulates the axon growth of spinal cord motor neurons in vitro These findings indicate that the CXCL12α-CXCR4 axis plays an important role in the regeneration of the neuromuscular junction after motor axon injury. The present results have important implications in the effort to find therapeutics and protocols to improve recovery of function after different forms of motor axon terminal damage. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

  12. Neuregulin1 displayed on motor axons regulates terminal Schwann cell-mediated synapse elimination at developing neuromuscular junctions.

    Science.gov (United States)

    Lee, Young Il; Li, Yue; Mikesh, Michelle; Smith, Ian; Nave, Klaus-Armin; Schwab, Markus H; Thompson, Wesley J

    2016-01-26

    Synaptic connections in the nervous system are rearranged during development and in adulthood as a feature of growth, plasticity, aging, and disease. Glia are implicated as active participants in these changes. Here we investigated a signal that controls the participation of peripheral glia, the terminal Schwann cells (SCs), at the neuromuscular junction (NMJ) in mice. Transgenic manipulation of the levels of membrane-tethered neuregulin1 (NRG1-III), a potent activator of SCs normally presented on motor axons, alters the rate of loss of motor inputs at NMJs during developmental synapse elimination. In addition, NMJs of adult transgenic mice that expressed excess axonal NRG1-III exhibited continued remodeling, in contrast to the more stable morphologies of controls. In fact, synaptic SCs of these adult mice with NRG1-III overexpression exhibited behaviors evident in wild type neonates during synapse elimination, including an affinity for the postsynaptic myofiber surface and phagocytosis of nerve terminals. Given that levels of NRG1-III expression normally peak during the period of synapse elimination, our findings identify axon-tethered NRG1 as a molecular determinant for SC-driven neuromuscular synaptic plasticity.

  13. Phosphorylation dynamics during early differentiation of human embryonic stem cells

    DEFF Research Database (Denmark)

    Van Hoof, Dennis; Muñoz, Javier; Braam, Stefan R

    2009-01-01

    Pluripotent stem cells self-renew indefinitely and possess characteristic protein-protein networks that remodel during differentiation. How this occurs is poorly understood. Using quantitative mass spectrometry, we analyzed the (phospho)proteome of human embryonic stem cells (hESCs) during...

  14. Phosphorylation dynamics during early differentiation of human embryonic stem cells

    NARCIS (Netherlands)

    van Hoof, D.; Munoz, J.; Braam, S.R.; Pinkse, M.W.H.; Linding, R.; Heck, A.J.R.; Mummery, C.L.; Krijgsveld, J.

    2009-01-01

    Pluripotent stem cells self-renew indefinitely and possess characteristic protein-protein networks that remodel during differentiation. How this occurs is poorly understood. Using quantitative mass spectrometry, we analyzed the (phospho)proteome of human embryonic stem cells (hESCs) during

  15. Improved genetic manipulation of human embryonic stem cells.

    NARCIS (Netherlands)

    Braam, S.R.; Denning, C.; van den Brink, S.; Kats, P.; Hochstenbach, R.; Passier, R.; Mummery, C.L.

    2008-01-01

    Low efficiency of transfection limits the ability to genetically manipulate human embryonic stem cells (hESCs), and differences in cell derivation and culture methods require optimization of transfection protocols. We transiently transferred multiple independent hESC lines with different growth

  16. Impact of 2-bromopropane on mouse embryonic stem cells and ...

    African Journals Online (AJOL)

    This study shows that 2-BP (5 to 10 μM) induces apoptotic processes in mouse embryonic stem cells (ESC-B5), but exerts no effects at treatment dosages below 5 μM. In ESC-B5 cells, 2-BP directly increased the content of reactive oxygen species (ROS), significantly increased the cytoplasmic free calcium and nitric oxide ...

  17. In vitro differentiation of mouse embryonic stem cells into functional ...

    African Journals Online (AJOL)

    Jane

    2011-08-22

    Aug 22, 2011 ... Key words: Embryonic stem cells, hepatic-like cells, in vitro differentiation, sodium butyrate, hepatocyte growth factor, dexamethason. INTRODUCTION. The liver is the major organ that provides multiple metabolic functions critical for the maintenance of homeostasis. One of the major causes of morbidity and.

  18. The ethics of patenting human embryonic stem cells.

    Science.gov (United States)

    Chapman, Audrey R

    2009-09-01

    Just as human embryonic stem cell research has generated controversy about the uses of human embryos for research and therapeutic applications, human embryonic stem cell patents raise fundamental ethical issues. The United States Patent and Trademark Office has granted foundational patents, including a composition of matter (or product) patent to the Wisconsin Alumni Research Foundation (WARF), the University of Wisconsin-Madison's intellectual property office. In contrast, the European Patent Office rejected the same WARF patent application for ethical reasons. This article assesses the appropriateness of these patents placing the discussion in the context of the deontological and consequentialist ethical issues related to human embryonic stem cell patenting. It advocates for a patent system that explicitly takes ethical factors into account and explores options for new types of intellectual property arrangements consistent with ethical concerns.

  19. PACAP interacts with PAC1 receptors to induce tissue plasminogen activator (tPA) expression and activity in schwann cell-like cultures.

    Science.gov (United States)

    Castorina, Alessandro; Waschek, James A; Marzagalli, Rubina; Cardile, Venera; Drago, Filippo

    2015-01-01

    Regeneration of peripheral nerves depends on the abilities of rejuvenating axons to migrate at the injury site through cellular debris and altered extracellular matrix, and then grow along the residual distal nerve sheath conduit and reinnervate synaptic targets. Considerable evidence suggest that glial cells participate in this process, although the mechanisms remain to be clarified. In cell culture, regenerating neurites secrete PACAP, a peptide shown to induce the expression of the protease tissue plasminogen activator (tPA) in neural cell types. In the present studies, we tested the hypothesis that PACAP can stimulate peripheral glial cells to produce tPA. More specifically, we addressed whether or not PACAP promoted the expression and activity of tPA in the Schwann cell line RT4-D6P2T, which shares biochemical and physical properties with Schwann cells. We found that PACAP dose- and time-dependently stimulated tPA expression both at the mRNA and protein level. Such effect was mimicked by maxadilan, a potent PAC1 receptor agonist, but not by the PACAP-related homolog VIP, suggesting a PAC1-mediated function. These actions appeared to be mediated at least in part by the Akt/CREB signaling cascade because wortmannin, a PI3K inhibitor, prevented peptide-driven CREB phosphorylation and tPA increase. Interestingly, treatment with BDNF mimicked PACAP actions on tPA, but acted through both the Akt and MAPK signaling pathways, while causing a robust increase in PACAP and PAC1 expression. PACAP6-38 totally blocked PACAP-driven tPA expression and in part hampered BDNF-mediated effects. We conclude that PACAP, acting through PAC1 receptors, stimulates tPA expression and activity in a Akt/CREB-dependent manner to promote proteolytic activity in Schwann-cell like cultures.

  20. PACAP interacts with PAC1 receptors to induce tissue plasminogen activator (tPA expression and activity in schwann cell-like cultures.

    Directory of Open Access Journals (Sweden)

    Alessandro Castorina

    Full Text Available Regeneration of peripheral nerves depends on the abilities of rejuvenating axons to migrate at the injury site through cellular debris and altered extracellular matrix, and then grow along the residual distal nerve sheath conduit and reinnervate synaptic targets. Considerable evidence suggest that glial cells participate in this process, although the mechanisms remain to be clarified. In cell culture, regenerating neurites secrete PACAP, a peptide shown to induce the expression of the protease tissue plasminogen activator (tPA in neural cell types. In the present studies, we tested the hypothesis that PACAP can stimulate peripheral glial cells to produce tPA. More specifically, we addressed whether or not PACAP promoted the expression and activity of tPA in the Schwann cell line RT4-D6P2T, which shares biochemical and physical properties with Schwann cells. We found that PACAP dose- and time-dependently stimulated tPA expression both at the mRNA and protein level. Such effect was mimicked by maxadilan, a potent PAC1 receptor agonist, but not by the PACAP-related homolog VIP, suggesting a PAC1-mediated function. These actions appeared to be mediated at least in part by the Akt/CREB signaling cascade because wortmannin, a PI3K inhibitor, prevented peptide-driven CREB phosphorylation and tPA increase. Interestingly, treatment with BDNF mimicked PACAP actions on tPA, but acted through both the Akt and MAPK signaling pathways, while causing a robust increase in PACAP and PAC1 expression. PACAP6-38 totally blocked PACAP-driven tPA expression and in part hampered BDNF-mediated effects. We conclude that PACAP, acting through PAC1 receptors, stimulates tPA expression and activity in a Akt/CREB-dependent manner to promote proteolytic activity in Schwann-cell like cultures.

  1. Behaviour of oligodendrocytes and Schwann cells in an experimental model of toxic demyelination of the central nervous system Comportamento de oligodendrócitos e células de Schwann em modelo experimental de desmielinização tóxica do sistema nervoso central

    Directory of Open Access Journals (Sweden)

    Dominguita Lühers Graça

    2001-06-01

    Full Text Available Oligodendrocytes and Schwann cells are engaged in myelin production, maintenance and repairing respectively in the central nervous system (CNS and the peripheral nervous system (PNS. Whereas oligodendrocytes act only within the CNS, Schwann cells are able to invade the CNS in order to make new myelin sheaths around demyelinated axons. Both cells have some limitations in their activities, i.e. oligodendrocytes are post-mitotic cells and Schwann cells only get into the CNS in the absence of astrocytes. Ethidium bromide (EB is a gliotoxic chemical that when injected locally within the CNS, induce demyelination. In the EB model of demyelination, glial cells are destroyed early after intoxication and Schwann cells are free to approach the naked central axons. In normal Wistar rats, regeneration of lost myelin sheaths can be achieved as early as thirteen days after intoxication; in Wistar rats immunosuppressed with cyclophosphamide the process is delayed and in rats administered cyclosporine it may be accelerated. Aiming the enlightening of those complex processes, all events concerning the myelinating cells in an experimental model are herein presented and discussed.Oligodendrócitos e células de Schwann realizam a produção e manutenção das bainhas de mielina, respectivamente no sistema nervoso central (SNC e periférico (SNP. As células de Schwann, à diferença dos oligodendrócitos, são capazes de invadir o SNC para remielinizar axônios desmielinizados, sempre que os astrócitos tenham sido destruídos. O brometo de etídio é uma droga gliotóxica usada para induzir desmielinização com o desaparecimento precoce de astrócitos, de modo que as células de Schwann têm liberdade para invadir o SNC. Em ratos Wistar normais, a remielinização é detectada treze dias após desmielinização; em ratos Wistar imunossuprimidos com ciclofosfamida a reparação do tecido é tardia, enquanto que em animais tratados com ciclosporina ela

  2. Differentiation of Adipocytes in Monolayer from Mouse Embryonic Stem Cells.

    Science.gov (United States)

    Cuaranta-Monroy, Ixchelt; Simandi, Zoltan; Nagy, Laszlo

    2016-01-01

    Obesity and its comorbidity incidence have increased worldwide during the past 10 years. In consequence, researchers have drawn their attention to the understanding of adipocyte differentiation. Several cellular model systems have been established; however no efficient protocol could be developed so far to differentiate the pluripotent embryonic stem cells to adipocytes. In this chapter, we describe a detailed protocol that is optimized for mouse embryonic stem cells. The result of this differentiation is a homogenous adipocyte monolayer culture that can be used for several applications including developmental and pharmacological research.

  3. Differentiation of Mouse Embryonic Stem Cells into Ventral Foregut Precursors

    DEFF Research Database (Denmark)

    Rothová, Michaela; Hölzenspies, Jurriaan J; Livigni, Alessandra

    2016-01-01

    Anterior definitive endoderm (ADE), the ventral foregut precursor, is both an important embryonic signaling center and a unique multipotent precursor of liver, pancreas, and other organs. Here, a method is described for the differentiation of mouse embryonic stem cells (mESCs) to definitive...... endoderm with pronounced anterior character. ADE-containing cultures can be produced in vitro by suspension (embryoid body) culture or in a serum-free adherent monolayer culture. ESC-derived ADE cells are committed to endodermal fates and can undergo further differentiation in vitro towards ventral foregut...

  4. Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth

    Energy Technology Data Exchange (ETDEWEB)

    Varga, Nora [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary); Vereb, Zoltan; Rajnavoelgyi, Eva [Department of Immunology, Medical and Health Science Centre, University of Debrecen, Debrecen (Hungary); Nemet, Katalin; Uher, Ferenc; Sarkadi, Balazs [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary); Apati, Agota, E-mail: apati@kkk.org.hu [Membrane Research Group of the Hungarian Academy of Sciences, Semmelweis University, Budapest (Hungary)

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer MSC like cells were derived from hESC by a simple and reproducible method. Black-Right-Pointing-Pointer Differentiation and immunosuppressive features of MSCl cells were similar to bmMSC. Black-Right-Pointing-Pointer MSCl cells as feeder cells support the undifferentiated growth of hESC. -- Abstract: Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could be propagated without changes in morphological or functional characteristics for more than 15 passages. These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the growth of undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures.

  5. The cell cycle as a brake for ?-cell regeneration from embryonic stem cells

    OpenAIRE

    El-Badawy, Ahmed; El-Badri, Nagwa

    2016-01-01

    The generation of insulin-producing ? cells from stem cells in vitro provides a promising source of cells for cell transplantation therapy in diabetes. However, insulin-producing cells generated from human stem cells show deficiency in many functional characteristics compared with pancreatic ? cells. Recent reports have shown molecular ties between the cell cycle and the differentiation mechanism of embryonic stem (ES) cells, assuming that cell fate decisions are controlled by the cell cycle ...

  6. Reprogramming Malignant Cancer Cells toward a Benign Phenotype following Exposure to Human Embryonic Stem Cell Microenvironment

    Science.gov (United States)

    Arena, Vincenzo; Arena, Manuel; Arena, Goffredo Orazio

    2017-01-01

    The embryonic microenvironment is well known to be non-permissive for tumor development because early developmental signals naturally suppress the expression of proto-oncogenes. In an analogous manner, mimicking an early embryonic environment during embryonic stem cell culture has been shown to suppress oncogenic phenotypes of cancer cells. Exosomes derived from human embryonic stem cells harbor substances that mirror the content of the cells of origin and have been reported to reprogram hematopoietic stem/progenitor cells via horizontal transfer of mRNA and proteins. However, the possibility that these embryonic stem cells-derived exosomes might be the main effectors of the anti-tumor effect mediated by the embryonic stem cells has not been explored yet. The present study aims to investigate whether exosomes derived from human embryonic stem cells can reprogram malignant cancer cells to a benign stage and reduce their tumorigenicity. We show that the embryonic stem cell-conditioned medium contains factors that inhibit cancer cell growth and tumorigenicity in vitro and in vivo. Moreover, we demonstrate that exosomes derived from human embryonic stem cells display anti-proliferation and pro-apoptotic effects, and decrease tumor size in a xenograft model. These exosomes are also able to transfer their cargo into target cancer cells, inducing a dose-dependent increase in SOX2, OCT4 and Nanog proteins, leading to a dose-dependent decrease of cancer cell growth and tumorigenicity. This study shows for the first time that human embryonic stem cell-derived exosomes play an important role in the tumor suppressive activity displayed by human embryonic stem cells. PMID:28068409

  7. Basic fibroblast growth factor is a key factor that induces bone marrow mesenchymal stem cells towards cells with Schwann cell phenotype.

    Science.gov (United States)

    Zhu, Hui; Yang, Aizhen; Du, Jinfeng; Li, Donghui; Liu, Mei; Ding, Fei; Gu, Xiaosong; Liu, Yan

    2014-01-24

    Bone marrow mesenchymal stem cells (MSCs) can be differentiate towards a Schwann cells (SCs) lineage when exposed to pre-inducing reagents β-mercaptoethanol (BME) and retinoic acid (RA), followed by inducing factors: forskolin (FSK), basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF), and heregulin (HRG). However, the underlying mechanisms remain unclear. Here, we investigated the individual effects of these inducing factors on the differentiation of MSCs towards SC phenotype in rats. We show that the omission of either HRG or PDGF from the induction medium is not sufficient to change the SC-like phenotype or the expression level of the SC marker, S100β. However, the omission of bFGF from the induction medium effectively blocked neural induction of the MSCs. Moreover, only bFGF was found to inhibit MSC proliferation during differentiation. To clarify the mechanism responsible for the effect of bFGF, we also investigated the activation of the extracellular signal-regulated kinase (ERK) pathway in the induced cells. Our results suggest that morphological changes in MSCs induced by bFGF depend on the activation of ERK, and bFGF may be an indispensable factor that induces MSCs to differentiate into cells with SCs phenotype. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  8. Human primordial germ cells migrate along nerve fibers and Schwann cells from the dorsal hind gut mesentery to the gonadal ridge

    DEFF Research Database (Denmark)

    Møllgård, Kjeld; Jespersen, Åse; Lutterodt, Melissa Catherine

    2010-01-01

    The aim of this study was to investigate the spatiotemporal development of autonomic nerve fibers and primordial germ cells (PGCs) along their migratory route from the dorsal mesentery to the gonadal ridges in human embryos using immunohistochemical markers and electron microscopy. Autonomic nerve...... and their intimate contact with PGCs. PGCs expressed GAGE, MAGE-A4, OCT4 and c-Kit. Serial paraffin sections showed that most PGCs were located inside bundles of autonomic nerve fibers with the majority adjacent to the most peripheral fibers (close to Schwann cells). We also show that both nerve fibers and PGCs...... arrive at the gonadal ridge between 29 and 33 days pc. In conclusion, our data suggest that PGCs in human embryos preferentially migrate along autonomic nerve fibers from the dorsal mesentery to the developing gonad where they are delivered via a fine nerve plexus....

  9. Proliferation- and migration-enhancing effects of ginseng and ginsenoside Rg1 through IGF-I- and FGF-2-signaling pathways on RSC96 Schwann cells.

    Science.gov (United States)

    Lu, Ming-Chin; Lai, Tung-Yuan; Hwang, Jin-Ming; Chen, Hsien-Te; Chang, Sheng-Huang; Tsai, Fuu-Jen; Wang, Hwai-Lee; Lin, Chien-Chung; Kuo, Wei-Wen; Huang, Chih-Yang

    2009-06-01

    The aim of the present study is to evaluate the proliferation- and migration-enhancing effects of ginseng and its component, ginsenoside (Rg1) on RSC96 Schwann cells. We investigated the molecular signaling pathways, which include: (1) survival signaling, IGFs-IGFIR-Akt-Bcl2 and proliferative signaling, cell cycle factors and mitogen-activated protein kinase (MAPK) pathways, (2) migrating and anti-scar signaling, FGF-2-uPA-MMPs.We treated RSC96 cells with different concentrations (100, 200, 300, 400, 500 microg ml(-1)) of ginseng and its constituent, Rg1 (5, 10, 15, 20, 25 microg ml(-1)). We observed a proliferative effect in a dose-dependent manner by PCNA western blotting assay, MTT assay, and wound healing test. Furthermore, we also found in the results of western blotting assay, ginseng and Rg1 enhance protein expression of IGF-I pathway regulators, cell cycle controlling proteins, and MAPK signaling pathways to promote the cell proliferation. In addition, ginseng and Rg1 also stimulated the FGF-2-uPA-MMP 9 migrating pathway to enhance the migration of RSC96 Schwann cells. Using MAPK chemical inhibitors, U0126, SB203580, and SP600125, the proliferative effects of ginseng and Rg1 on RSC96 cells were identified to be MAPK signaling-dependent. On the basis of the results, applying appropriate doses of ginseng and Rg1 with biomedical materials would be a potential approach for enhancing neuron regeneration. 2009 John Wiley & Sons, Ltd.

  10. Growth inhibition of mouse embryonic stem (ES) cells on the feeders ...

    African Journals Online (AJOL)

    Mouse embryonic stem cells (mESCs) can be propagated in vitro on the feeders of mouse embryonic fibroblasts. In this study, we found growth inhibition of mESCs cultured on embryonic fibroblast feeders derived from different livestock animals. Under the same condition, mESCs derived from mouse embryonic fibroblast ...

  11. File list: Oth.Emb.10.AllAg.Embryonic_germ_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.10.AllAg.Embryonic_germ_cells mm9 TFs and others Embryo Embryonic germ cell...s http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Emb.10.AllAg.Embryonic_germ_cells.bed ...

  12. File list: Oth.Emb.05.AllAg.Embryonic_germ_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.05.AllAg.Embryonic_germ_cells mm9 TFs and others Embryo Embryonic germ cell...s http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Emb.05.AllAg.Embryonic_germ_cells.bed ...

  13. File list: Pol.Emb.20.AllAg.Embryonic_germ_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Emb.20.AllAg.Embryonic_germ_cells mm9 RNA polymerase Embryo Embryonic germ cell...s http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Emb.20.AllAg.Embryonic_germ_cells.bed ...

  14. File list: Oth.Emb.50.AllAg.Embryonic_germ_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.50.AllAg.Embryonic_germ_cells mm9 TFs and others Embryo Embryonic germ cell...s http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Emb.50.AllAg.Embryonic_germ_cells.bed ...

  15. File list: Pol.Emb.10.AllAg.Embryonic_germ_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Emb.10.AllAg.Embryonic_germ_cells mm9 RNA polymerase Embryo Embryonic germ cell...s http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Emb.10.AllAg.Embryonic_germ_cells.bed ...

  16. File list: Pol.Emb.05.AllAg.Embryonic_germ_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Emb.05.AllAg.Embryonic_germ_cells mm9 RNA polymerase Embryo Embryonic germ cell...s http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Emb.05.AllAg.Embryonic_germ_cells.bed ...

  17. File list: Pol.Emb.50.AllAg.Embryonic_germ_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Emb.50.AllAg.Embryonic_germ_cells mm9 RNA polymerase Embryo Embryonic germ cell...s http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Emb.50.AllAg.Embryonic_germ_cells.bed ...

  18. File list: Oth.Emb.20.AllAg.Embryonic_germ_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.20.AllAg.Embryonic_germ_cells mm9 TFs and others Embryo Embryonic germ cell...s http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Emb.20.AllAg.Embryonic_germ_cells.bed ...

  19. Enhanced noradrenergic axon regeneration into schwann cell-filled PVDF-TrFE conduits after complete spinal cord transection.

    Science.gov (United States)

    Lee, Yee-Shuan; Wu, Siliang; Arinzeh, Treena Livingston; Bunge, Mary Bartlett

    2017-02-01

    Schwann cell (SC) transplantation has been utilized for spinal cord repair and demonstrated to be a promising therapeutic strategy. In this study, we investigated the feasibility of combining SC transplantation with novel conduits to bridge the completely transected adult rat spinal cord. This is the first and initial study to evaluate the potential of using a fibrous piezoelectric polyvinylidene fluoride trifluoroethylene (PVDF-TrFE) conduit with SCs for spinal cord repair. PVDF-TrFE has been shown to enhance neurite growth in vitro and peripheral nerve repair in vivo. In this study, SCs adhered and proliferated when seeded onto PVDF-TrFE scaffolds in vitro. SCs and PVDF-TrFE conduits, consisting of random or aligned fibrous inner walls, were transplanted into transected rat spinal cords for 3 weeks to examine early repair. Glial fibrillary acidic protein (GFAP)+ astrocyte processes and GFP (green fluorescent protein)-SCs were interdigitated at both rostral and caudal spinal cord/SC transplant interfaces in both types of conduits, indicative of permissivity to axon growth. More noradrenergic/DβH+ (dopamine-beta-hydroxylase) brainstem axons regenerated across the transplant when greater numbers of GFAP+ astrocyte processes were present. Aligned conduits promoted extension of DβH+ axons and GFAP+ processes farther into the transplant than random conduits. Sensory CGRP+ (calcitonin gene-related peptide) axons were present at the caudal interface. Blood vessels formed throughout the transplant in both conduits. This study demonstrates that PVDF-TrFE conduits harboring SCs are promising for spinal cord repair and deserve further investigation. Biotechnol. Bioeng. 2017;114: 444-456. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  20. Derivation of human embryonic stem cells in defined conditions.

    Science.gov (United States)

    Ludwig, Tenneille E; Levenstein, Mark E; Jones, Jeffrey M; Berggren, W Travis; Mitchen, Erika R; Frane, Jennifer L; Crandall, Leann J; Daigh, Christine A; Conard, Kevin R; Piekarczyk, Marian S; Llanas, Rachel A; Thomson, James A

    2006-02-01

    We have previously reported that high concentrations of basic fibroblast growth factor (bFGF) support feeder-independent growth of human embryonic stem (ES) cells, but those conditions included poorly defined serum and matrix components. Here we report feeder-independent human ES cell culture that includes protein components solely derived from recombinant sources or purified from human material. We describe the derivation of two new human ES cell lines in these defined culture conditions.

  1. Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay

    Science.gov (United States)

    The Embryonic Stem Cell Test (EST) is an assay which evaluates xenobiotic-induced effects using three endpoints: mouse embryonic stem cell (mESC) differentiation, mESC viability, and 3T3-cell viability. Our research goal was to develop an improved high-throughput assay by establi...

  2. In vitro differentiation of mouse embryonic stem cells into functional ...

    African Journals Online (AJOL)

    In vitro differentiation of mouse embryonic stem cells into functional hepatocytes by sodium butyrate, hepatocyte growth factor and dexamethasone under ... under chemically defined conditions, which might be useful as an in vitro system for hepatocyte transplantation therapy and toxicity screening in drug discovery.

  3. Gene targeting in embryonic stem cells, II: conditional technologies

    Science.gov (United States)

    Genome modification via transgenesis has allowed researchers to link genotype and phenotype as an alternative approach to the characterization of random mutations through evolution. The synergy of technologies from the fields of embryonic stem (ES) cells, gene knockouts, and protein-mediated recombi...

  4. Human embryonic stem cell transplantation to repair the infarcted myocardium

    National Research Council Canada - National Science Library

    Leor, Jonathan; Gerecht, Sharon; Cohen, Smadar; Miller, Liron; Holbova, Radka; Ziskind, Anna; Shachar, Michal; Feinberg, Micha S; Guetta, Esther; Itskovitz-Eldor, Joseph

    2007-01-01

    To test the hypothesis that human embryonic stem cells (hESCs) can be guided to form new myocardium by transplantation into the normal or infarcted heart, and to assess the influence of hESC-derived cardiomyocytes (hESCMs...

  5. Primordial germ cell specification from embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Wei Wei

    Full Text Available BACKGROUND: Primordial germ cell (PGC specification is the first crucial step in germ line development. However, owing to significant challenges regarding the in vivo system, such as the complex cellular environment and potential problems with embryo manipulation, it is desirable to generate embryonic stem (ES cells that are capable of overcoming these aforementioned limitations in order to provide a potential in vitro model to recapitulate the developmental processes in vivo. METHODOLOGY AND PRINCIPAL FINDINGS: Here, we studied the detailed process of PGC specification from stella-GFP ES cells. We first observed the heterogeneous expression of stella in ES cells. However, neither Stella-positive ES cells nor Stella-negative ES cells shared a similar gene expression pattern with either PGCs or PGC precursors. Second, we derived PGCs from ES cells using two differentiation methods, namely the attachment culture technique and the embryoid body (EB method. Compared with PGCs derived via the attachment culture technique, PGCs derived via the EB method that had undergone the sequential erasure of Peg3 followed by Igf2r resulted in a cell line in which the expression dynamics of T, Fgf8 and Sox17, in addition to the expression of the epiblast markers, were more similar to the in vivo expression, thus demonstrating that the process of PGC derivation was more faithfully recapitulated using the EB method. Furthermore, we developed an in vitro model of PGC specification in a completely chemically defined medium (CDM that indicated that BMP4 and Wnt3a promoted PGC derivation, whereas BMP8b and activinA had no observable effect on PGC derivation. CONCLUSIONS AND SIGNIFICANCE: The in vitro model we have established can recapitulate the developmental processes in vivo and provides new insights into the mechanism of PGC specification.

  6. File list: DNS.Emb.20.AllAg.Embryonic_germ_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Emb.20.AllAg.Embryonic_germ_cells mm9 DNase-seq Embryo Embryonic germ cells htt...p://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/DNS.Emb.20.AllAg.Embryonic_germ_cells.bed ...

  7. File list: Unc.Emb.50.AllAg.Embryonic_germ_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Emb.50.AllAg.Embryonic_germ_cells mm9 Unclassified Embryo Embryonic germ cells ...SRX761312,SRX761313 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.Emb.50.AllAg.Embryonic_germ_cells.bed ...

  8. File list: DNS.Emb.10.AllAg.Embryonic_germ_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Emb.10.AllAg.Embryonic_germ_cells mm9 DNase-seq Embryo Embryonic germ cells htt...p://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/DNS.Emb.10.AllAg.Embryonic_germ_cells.bed ...

  9. File list: Unc.Emb.10.AllAg.Embryonic_germ_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Emb.10.AllAg.Embryonic_germ_cells mm9 Unclassified Embryo Embryonic germ cells ...SRX761312,SRX761313 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.Emb.10.AllAg.Embryonic_germ_cells.bed ...

  10. File list: His.Emb.10.AllAg.Embryonic_germ_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Emb.10.AllAg.Embryonic_germ_cells mm9 Histone Embryo Embryonic germ cells SRX27...p://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Emb.10.AllAg.Embryonic_germ_cells.bed ...

  11. File list: ALL.Emb.20.AllAg.Embryonic_germ_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Emb.20.AllAg.Embryonic_germ_cells mm9 All antigens Embryo Embryonic germ cells ...62,SRX685754,SRX685760,SRX336513,SRX336524,SRX685756,SRX685755,SRX685761,SRX336255 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.20.AllAg.Embryonic_germ_cells.bed ...

  12. File list: DNS.Emb.50.AllAg.Embryonic_germ_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Emb.50.AllAg.Embryonic_germ_cells mm9 DNase-seq Embryo Embryonic germ cells htt...p://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/DNS.Emb.50.AllAg.Embryonic_germ_cells.bed ...

  13. File list: ALL.Emb.10.AllAg.Embryonic_germ_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Emb.10.AllAg.Embryonic_germ_cells mm9 All antigens Embryo Embryonic germ cells ...24,SRX685755,SRX685756,SRX685757,SRX685758,SRX685761,SRX685762,SRX685760,SRX336255 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.10.AllAg.Embryonic_germ_cells.bed ...

  14. File list: ALL.Emb.05.AllAg.Embryonic_germ_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Emb.05.AllAg.Embryonic_germ_cells mm9 All antigens Embryo Embryonic germ cells ...65,SRX335426,SRX336256,SRX336475,SRX272024,SRX336255,SRX685757,SRX685754,SRX685762 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.05.AllAg.Embryonic_germ_cells.bed ...

  15. File list: Unc.Emb.20.AllAg.Embryonic_germ_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Unc.Emb.20.AllAg.Embryonic_germ_cells mm9 Unclassified Embryo Embryonic germ cells ...SRX761312,SRX761313 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Unc.Emb.20.AllAg.Embryonic_germ_cells.bed ...

  16. File list: His.Emb.20.AllAg.Embryonic_germ_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Emb.20.AllAg.Embryonic_germ_cells mm9 Histone Embryo Embryonic germ cells SRX27...p://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Emb.20.AllAg.Embryonic_germ_cells.bed ...

  17. File list: ALL.Emb.50.AllAg.Embryonic_germ_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Emb.50.AllAg.Embryonic_germ_cells mm9 All antigens Embryo Embryonic germ cells ...59,SRX685762,SRX685761,SRX685754,SRX685757,SRX685760,SRX335426,SRX761313,SRX336255 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Emb.50.AllAg.Embryonic_germ_cells.bed ...

  18. File list: His.Emb.05.AllAg.Embryonic_germ_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Emb.05.AllAg.Embryonic_germ_cells mm9 Histone Embryo Embryonic germ cells SRX68...p://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Emb.05.AllAg.Embryonic_germ_cells.bed ...

  19. File list: DNS.Emb.05.AllAg.Embryonic_germ_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Emb.05.AllAg.Embryonic_germ_cells mm9 DNase-seq Embryo Embryonic germ cells htt...p://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/DNS.Emb.05.AllAg.Embryonic_germ_cells.bed ...

  20. File list: His.Emb.50.AllAg.Embryonic_germ_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available His.Emb.50.AllAg.Embryonic_germ_cells mm9 Histone Embryo Embryonic germ cells SRX27...p://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/His.Emb.50.AllAg.Embryonic_germ_cells.bed ...

  1. Polycomb enables primitive endoderm lineage priming in embryonic stem cells

    DEFF Research Database (Denmark)

    Illingworth, Robert S; Hölzenspies, Jurriaan J; Roske, Fabian V

    2016-01-01

    Mouse embryonic stem cells (ESCs), like the blastocyst from which they are derived, contain precursors of the epiblast (Epi) and primitive endoderm (PrEn) lineages. While transient in vivo, these precursor populations readily interconvert in vitro. We show that altered transcription is the driver...... polycomb with dynamic changes in transcription and stalled lineage commitment, allowing cells to explore alternative choices prior to a definitive decision....

  2. [Progress in the research of germ cell from human embryonic stem cells].

    Science.gov (United States)

    Guo, Xin; Cai, Zhi-Ming; Gui, Yao-Ting

    2006-01-01

    As the development of spontaneous differentiation of germ cells and gametogenesis from mouse embryonic stem cells (mES) in vitro, hES (human embryonic stem cells) also have potential to differentiated into germ cells in theory. This review focuses on the stem cells niches and genes regulating the hES differentiation toward germ cells, as well as the recent advance and application on the reproductive medicine and therapy of infertility.

  3. A practical guide for the identification of membrane and plasma membrane proteins in human embryonic stem cells and human embryonal carcinoma cells.

    NARCIS (Netherlands)

    Dormeyer, W.; van Hoof, D.; Mummery, C.L.; Krijgsveld, J.; Heck, A.

    2008-01-01

    The identification of (plasma) membrane proteins in cells can provide valuable insights into the regulation of their biological processes. Pluripotent cells such as human embryonic stem cells and embryonal carcinoma cells are capable of unlimited self-renewal and share many of the biological

  4. A Combination Tissue Engineering Strategy for Schwann Cell-Induced Spinal Cord Repair

    Science.gov (United States)

    2016-10-01

    evaporated before using for cell culture studies. For in vitro experiments, the scaffolds were cut into 6mm disks and sterilized by ultraviolet ...of the PVDF-TrFE conduit in situ to understand if and how piezoelectricity may contribute to trans- planted SC distribution and axon regeneration. In...Barry J, Vertegel A, Ray S, et al. Spinal Cord Injury: A Review of Current Therapy, Future Treatments, and Basic Science Frontiers. Neurochem Res

  5. Viscoelastic and dynamic properties of embryonic stem cells

    DEFF Research Database (Denmark)

    Ritter, Christine

    ofthe cells themselves. In this thesis, the viscoelastic properties of mouse embryonic stem cells primedeither toward the epiblast (Epi) or the primitive endoderm (PrE) lineage were investigated.Optical tweezers were used to measure the fluctuations of endogenous lipid granules and therebydraw...... diffusive process was determined to becontinuous time random walk (CTRW).Upon exciting pluripotency, changes occur in the nucleus of stem cells. Chromatin remodeling,the recruitment of lamin A to the nucleoskeleton and stiffening of the cells were reported changescaused by differentiation...

  6. The analgesic effect on neuropathic pain of retrogradely transported botulinum neurotoxin A involves Schwann cells and astrocytes.

    Directory of Open Access Journals (Sweden)

    Sara Marinelli

    Full Text Available In recent years a growing debate is about whether botulinum neurotoxins are retrogradely transported from the site of injection. Immunodetection of cleaved SNAP-25 (cl-SNAP-25, the protein of the SNARE complex targeted by botulinum neurotoxin serotype A (BoNT/A, could represent an excellent approach to investigate the mechanism of action on the nociceptive pathways at peripheral and/or central level. After peripheral administration of BoNT/A, we analyzed the expression of cl-SNAP-25, from the hindpaw's nerve endings to the spinal cord, together with the behavioral effects on neuropathic pain. We used the chronic constriction injury of the sciatic nerve in CD1 mice as animal model of neuropathic pain. We evaluated immunostaining of cl-SNAP-25 in the peripheral nerve endings, along the sciatic nerve, in dorsal root ganglia and in spinal dorsal horns after intraplantar injection of saline or BoNT/A, alone or colocalized with either glial fibrillar acidic protein, GFAP, or complement receptor 3/cluster of differentiation 11b, CD11b, or neuronal nuclei, NeuN, depending on the area investigated. Immunofluorescence analysis shows the presence of the cl-SNAP-25 in all tissues examined, from the peripheral endings to the spinal cord, suggesting a retrograde transport of BoNT/A. Moreover, we performed in vitro experiments to ascertain if BoNT/A was able to interact with the proliferative state of Schwann cells (SC. We found that BoNT/A modulates the proliferation of SC and inhibits the acetylcholine release from SC, evidencing a new biological effect of the toxin and further supporting the retrograde transport of the toxin along the nerve and its ability to influence regenerative processes. The present results strongly sustain a combinatorial action at peripheral and central neural levels and encourage the use of BoNT/A for the pathological pain conditions difficult to treat in clinical practice and dramatically impairing patients' quality of life.

  7. Nerve Regeneration Potential of Protocatechuic Acid in RSC96 Schwann Cells by Induction of Cellular Proliferation and Migration through IGF-IR-PI3K-Akt Signaling.

    Science.gov (United States)

    Ju, Da-Tong; Liao, Hung-En; Shibu, Marthandam Asokan; Ho, Tsung-Jung; Padma, Viswanadha Vijaya; Tsai, Fuu-Jen; Chung, Li-Chin; Day, Cecilia Hsuan; Lin, Chien-Chung; Huang, Chih-Yang

    2015-12-31

    Peripheral nerve injuries, caused by accidental trauma, acute compression or surgery, often result in temporary or life-long neuronal dysfunctions and inflict great economic or social burdens on the patients. Nerve cell proliferation is an essential process to restore injured nerves of adults. Schwann cells play a crucial role in endogenous repair of peripheral nerves due to their ability to proliferate, migrate and provide trophic support to axons via expression of various neurotrophic factors, such as the nerve growth factor (NGF), especially after nerve injury. Protocatechuic acid (PCA) is a dihydroxybenzoic acid, a type of phenolic acid, isolated from the kernels of Alpinia oxyphylla Miq (AOF), a traditional Chinese herbal medicine the fruits of which are widely used as a tonic, aphrodisiac, anti-salivation and anti-diarrheatic. This study investigated the molecular mechanisms by which PCA induces Schwann cell proliferation by activating IGF-IR-PI3K-Akt pathway. Treatment with PCA induces phosphorylation of the insulin-like growth factor-I (IGF-I)-mediated phosphatidylinositol 3 kinase/serine - threonine kinase (PI3K/Akt) pathway, and activates expression of cell nuclear antigen (PCNA) in a dose-dependent manner. Cell cycle analysis after 18 h of treatment showed that proliferation of the RSC96 cells was enhanced by PCA treatment. The PCA induced proliferation was accompanied by modulation in the expressions of cell cycle proteins cyclin D1, cyclin E and cyclin A. Knockdown of PI3K using small interfering RNA (siRNA) and inhibition of IGF-IR receptor resulted in the reduction in cell survival proteins. The results collectively showed that PCA treatment promoted cell proliferation and cell survival via IGF-I signaling.

  8. Abortion, embryonic stem cell research, and waste.

    Science.gov (United States)

    Jensen, David A

    2008-01-01

    Can one consistently deny the permissibility of abortion while endorsing the killing of human embryos for the sake of stem cell research? The question is not trivial; for even if one accepts that abortion is prima facie wrong in all cases, there are significant differences with many of the embryos used for stem cell research from those involved in abortion--most prominently, many have been abandoned in vitro, and appear to have no reasonably likely meaningful future. On these grounds one might think to maintain a strong position against abortion but endorse killing human embryos for the sake of stem cell research and its promising benefits. I will argue, however, that these differences are not decisive. Thus, one who accepts a strong view against abortion is committed to the moral impermissibility of killing human embryos for the sake of stem cell research. I do not argue for the moral standing of either abortion or the killing of embryos for stem cell research; I only argue for the relation between the two. Thus the conclusion is relevant to those with a strong view in favor of the permissibility of killing embryos for the sake of research as much as for those who may strongly oppose abortion; neither can consider their position in isolation from the other.

  9. The cell cycle as a brake for β-cell regeneration from embryonic stem cells.

    Science.gov (United States)

    El-Badawy, Ahmed; El-Badri, Nagwa

    2016-01-13

    The generation of insulin-producing β cells from stem cells in vitro provides a promising source of cells for cell transplantation therapy in diabetes. However, insulin-producing cells generated from human stem cells show deficiency in many functional characteristics compared with pancreatic β cells. Recent reports have shown molecular ties between the cell cycle and the differentiation mechanism of embryonic stem (ES) cells, assuming that cell fate decisions are controlled by the cell cycle machinery. Both β cells and ES cells possess unique cell cycle machinery yet with significant contrasts. In this review, we compare the cell cycle control mechanisms in both ES cells and β cells, and highlight the fundamental differences between pluripotent cells of embryonic origin and differentiated β cells. Through critical analysis of the differences of the cell cycle between these two cell types, we propose that the cell cycle of ES cells may act as a brake for β-cell regeneration. Based on these differences, we discuss the potential of modulating the cell cycle of ES cells for the large-scale generation of functionally mature β cells in vitro. Further understanding of the factors that modulate the ES cell cycle will lead to new approaches to enhance the production of functional mature insulin-producing cells, and yield a reliable system to generate bona fide β cells in vitro.

  10. Epigenetic stability, adaptability, and reversibility in human embryonic stem cells

    OpenAIRE

    Tompkins, Joshua D.; Hall, Christine; Chen, Vincent Chang-yi; Li, Arthur Xuejun; Wu, Xiwei; Hsu, David; Couture, Larry A.; Riggs, Arthur D.

    2012-01-01

    The stability of human embryonic stem cells (hESCs) is of critical importance for both experimental and clinical applications. We find that as an initial response to altered culture conditions, hESCs change their transcription profile for hundreds of genes and their DNA methylation profiles for several genes outside the core pluripotency network. After adaption to conditions of feeder-free defined and/or xeno-free culture systems, expression and DNA methylation profiles are quite stable for a...

  11. Generation of stomach tissue from mouse embryonic stem cells.

    Science.gov (United States)

    Noguchi, Taka-aki K; Ninomiya, Naoto; Sekine, Mari; Komazaki, Shinji; Wang, Pi-Chao; Asashima, Makoto; Kurisaki, Akira

    2015-08-01

    Successful pluripotent stem cell differentiation methods have been developed for several endoderm-derived cells, including hepatocytes, β-cells and intestinal cells. However, stomach lineage commitment from pluripotent stem cells has remained a challenge, and only antrum specification has been demonstrated. We established a method for stomach differentiation from embryonic stem cells by inducing mesenchymal Barx1, an essential gene for in vivo stomach specification from gut endoderm. Barx1-inducing culture conditions generated stomach primordium-like spheroids, which differentiated into mature stomach tissue cells in both the corpus and antrum by three-dimensional culture. This embryonic stem cell-derived stomach tissue (e-ST) shared a similar gene expression profile with adult stomach, and secreted pepsinogen as well as gastric acid. Furthermore, TGFA overexpression in e-ST caused hypertrophic mucus and gastric anacidity, which mimicked Ménétrier disease in vitro. Thus, in vitro stomach tissue derived from pluripotent stem cells mimics in vivo development and can be used for stomach disease models.

  12. Raman microscopy of individual living human embryonic stem cells

    DEFF Research Database (Denmark)

    Novikov, Sergey M.; Beermann, Jonas; Bozhevolnyi, Sergey I.

    2010-01-01

    We demonstrate the possibility of mapping the distribution of different biomolecules in living human embryonic stem cells grown on glass substrates, without the need for fluorescent markers. In our work we improve the quality of measurements by finding a buffer that gives low fluorescence, growing...... cells on glass substrates (whose Raman signals are relatively weak compared to that of the cells) and having the backside covered with gold to improve the image contrast under direct white light illumination. The experimental setup used for Raman microscopy is the commercially available confocal...

  13. Combination Therapy with c-Met and Src Inhibitors Induces Caspase-Dependent Apoptosis of Merlin-Deficient Schwann Cells and Suppresses Growth of Schwannoma Cells.

    Science.gov (United States)

    Fuse, Marisa A; Plati, Stephani Klingeman; Burns, Sarah S; Dinh, Christine T; Bracho, Olena; Yan, Denise; Mittal, Rahul; Shen, Rulong; Soulakova, Julia N; Copik, Alicja J; Liu, Xue Zhong; Telischi, Fred F; Chang, Long-Sheng; Franco, Maria Clara; Fernandez-Valle, Cristina

    2017-11-01

    Neurofibromatosis type 2 (NF2) is a nervous system tumor disorder caused by inactivation of the merlin tumor suppressor encoded by the NF2 gene. Bilateral vestibular schwannomas are a diagnostic hallmark of NF2. Mainstream treatment options for NF2-associated tumors have been limited to surgery and radiotherapy; however, off-label uses of targeted molecular therapies are becoming increasingly common. Here, we investigated drugs targeting two kinases activated in NF2-associated schwannomas, c-Met and Src. We demonstrated that merlin-deficient mouse Schwann cells (MD-MSC) treated with the c-Met inhibitor, cabozantinib, or the Src kinase inhibitors, dasatinib and saracatinib, underwent a G 1 cell-cycle arrest. However, when MD-MSCs were treated with a combination of cabozantinib and saracatinib, they exhibited caspase-dependent apoptosis. The combination therapy also significantly reduced growth of MD-MSCs in an orthotopic allograft mouse model by greater than 80% of vehicle. Moreover, human vestibular schwannoma cells with NF2 mutations had a 40% decrease in cell viability when treated with cabozantinib and saracatinib together compared with the vehicle control. This study demonstrates that simultaneous inhibition of c-Met and Src signaling in MD-MSCs triggers apoptosis and reveals vulnerable pathways that could be exploited to develop NF2 therapies. Mol Cancer Ther; 16(11); 2387-98. ©2017 AACR . ©2017 American Association for Cancer Research.

  14. The Glia Response after Peripheral Nerve Injury: A Comparison between Schwann Cells and Olfactory Ensheathing Cells and Their Uses for Neural Regenerative Therapies.

    Science.gov (United States)

    Barton, Matthew J; John, James St; Clarke, Mary; Wright, Alison; Ekberg, Jenny

    2017-01-29

    The peripheral nervous system (PNS) exhibits a much larger capacity for regeneration than the central nervous system (CNS). One reason for this difference is the difference in glial cell types between the two systems. PNS glia respond rapidly to nerve injury by clearing debris from the injury site, supplying essential growth factors and providing structural support; all of which enhances neuronal regeneration. Thus, transplantation of glial cells from the PNS is a very promising therapy for injuries to both the PNS and the CNS. There are two key types of PNS glia: olfactory ensheathing cells (OECs), which populate the olfactory nerve, and Schwann cells (SCs), which are present in the rest of the PNS. These two glial types share many similar morphological and functional characteristics but also exhibit key differences. The olfactory nerve is constantly turning over throughout life, which means OECs are continuously stimulating neural regeneration, whilst SCs only promote regeneration after direct injury to the PNS. This review presents a comparison between these two PNS systems in respect to normal physiology, developmental anatomy, glial functions and their responses to injury. A thorough understanding of the mechanisms and differences between the two systems is crucial for the development of future therapies using transplantation of peripheral glia to treat neural injuries and/or disease.

  15. Gene expression heterogeneities in embryonic stem cell populations

    DEFF Research Database (Denmark)

    Martinez Arias, Alfonso; Brickman, Joshua M

    2011-01-01

    Stem and progenitor cells are populations of cells that retain the capacity to populate specific lineages and to transit this capacity through cell division. However, attempts to define markers for stem cells have met with limited success. Here we consider whether this limited success reflects...... an intrinsic requirement for heterogeneity with stem cell populations. We focus on Embryonic Stem (ES) cells, in vitro derived cell lines from the early embryo that are considered both pluripotent (able to generate all the lineages of the future embryo) and indefinitely self renewing. We examine the relevance...... of recently reported heterogeneities in ES cells and whether these heterogeneities themselves are inherent requirements of functional potency and self renewal....

  16. From transplanting Schwann cells in experimental rat spinal cord injury to their transplantation into human injured spinal cord in clinical trials.

    Science.gov (United States)

    Bunge, Mary B; Monje, Paula V; Khan, Aisha; Wood, Patrick M

    2017-01-01

    Among the potential therapies designed to repair the injured spinal cord is cell transplantation, notably the use of autologous adult human Schwann cells (SCs). Here, we detail some of the critical research accomplished over the last four decades to establish a foundation that enables these cells to be tested in clinical trials. New culture systems allowed novel information to be gained about SCs, including discovering ways to stimulate their proliferation to acquire adequately large numbers for transplantation into the injured human spinal cord. Transplantation of rat SCs into rat models of spinal cord injury has demonstrated that SCs promote repair of injured spinal cord. Additional work required to gain approval from the Food and Drug Administration for the first SC trial in the Miami Project is disclosed. This trial and a second one now underway are described. © 2017 Elsevier B.V. All rights reserved.

  17. Chitosan Degradation Products Promote Nerve Regeneration by Stimulating Schwann Cell Proliferation via miR-27a/FOXO1 Axis.

    Science.gov (United States)

    Wang, Yongjun; Zhao, Yahong; Sun, Cheng; Hu, Wen; Zhao, Jing; Li, Guicai; Zhang, Luzhong; Liu, Mei; Liu, Yan; Ding, Fei; Yang, Yumin; Gu, Xiaosong

    2016-01-01

    Natural polysaccharides are biomaterials widely used for constructing scaffolds in tissue engineering. While natural polysaccharides have been shown to robustly promote tissue regeneration, the underlying molecular mechanism remains largely unknown. Here, we show that chitooligosaccharides (COS), the intermediate products of chitosan degradation, stimulate peripheral nerve regeneration in rats. Our experiment also shows that COS stimulate the proliferation of Schwann cells (SCs) during nerve regeneration. By analyzing the transcriptome and gene regulatory network, we identified the miR-27a/FOXO1 axis as the main signaling pathway for mediating the proliferative effects of COS on SCs. COS increase the expression level of miR-27a and cause a reduction of FOXO1, which subsequently accelerates the cell cycle and stimulates SC proliferation to stimulate nerve regeneration. These findings define a basic pathway for oligosaccharides-mediated cell proliferation and reveal a novel aspect of polysaccharide biomaterials in tissue engineering.

  18. Extracellular Matrix and Integrins in Embryonic Stem Cell Differentiation.

    Science.gov (United States)

    Wang, Han; Luo, Xie; Leighton, Jake

    2015-01-01

    Embryonic stem cells (ESCs) are pluripotent cells with great therapeutic potentials. The in vitro differentiation of ESC was designed by recapitulating embryogenesis. Significant progress has been made to improve the in vitro differentiation protocols by toning soluble maintenance factors. However, more robust methods for lineage-specific differentiation and maturation are still under development. Considering the complexity of in vivo embryogenesis environment, extracellular matrix (ECM) cues should be considered besides growth factor cues. ECM proteins bind to cells and act as ligands of integrin receptors on cell surfaces. Here, we summarize the role of the ECM and integrins in the formation of three germ layer progenies. Various ECM-integrin interactions were found, facilitating differentiation toward definitive endoderm, hepatocyte-like cells, pancreatic beta cells, early mesodermal progenitors, cardiomyocytes, neuroectoderm lineages, and epidermal cells, such as keratinocytes and melanocytes. In the future, ECM combinations for the optimal ESC differentiation environment will require substantial study.

  19. Genetic engineering of human embryonic stem cells with lentiviral vectors.

    Science.gov (United States)

    Xiong, Chen; Tang, Dong-Qi; Xie, Chang-Qing; Zhang, Li; Xu, Ke-Feng; Thompson, Winston E; Chou, Wayne; Gibbons, Gary H; Chang, Lung-Ji; Yang, Li-Jun; Chen, Yuqing E

    2005-08-01

    Human embryonic stem (hES) cells present a valuable source of cells with a vast therapeutic potential. However, the low efficiency of directed differentiation of hES cells remains a major obstacle in their uses for regenerative medicine. While differentiation may be controlled by the genetic manipulation, effective and efficient gene transfer into hES cells has been an elusive goal. Here, we show stable and efficient genetic manipulations of hES cells using lentiviral vectors. This method resulted in the establishment of stable gene expression without loss of pluripotency in hES cells. In addition, lentiviral vectors were effective in conveying the expression of an U6 promoter-driven small interfering RNA (siRNA), which was effective in silencing its specific target. Taken together, our results suggest that lentiviral gene delivery holds great promise for hES cell research and application.

  20. EDA-Containing Fibronectin Increases Proliferation of Embryonic Stem Cells

    Science.gov (United States)

    Losino, Noelia; Waisman, Ariel; Solari, Claudia; Luzzani, Carlos; Espinosa, Darío Fernández; Sassone, Alina; Muro, Andrés F.; Miriuka, Santiago; Sevlever, Gustavo; Barañao, Lino; Guberman, Alejandra

    2013-01-01

    Embryonic stem cells (ESC) need a set of specific factors to be propagated. They can also grow in conditioned medium (CM) derived from a bovine granulosa cell line BGC (BGC-CM), a medium that not only preserves their main features but also increases ESC´s proliferation rate. The mitogenic properties of this medium were previously reported, ascribing this effect to an alternative spliced generated fibronectin isoform that contains the extra domain A (FN EDA+). Here, we investigated if the FN EDA+ isoform increased proliferation of mouse and human ES cells. We analyzed cell proliferation using conditioned media produced by different mouse embryonic fibroblast (MEF) lines genetically engineered to express FN constitutively including or excluding the EDA domain (FN EDA-), and in media supplemented with recombinant peptides containing or not the EDA. We found that the presence of EDA in the medium increased mouse and human ESC’s proliferation rate. Here we showed for the first time that this FN isoform enhances ESC’s proliferation. These findings suggest a possible conserved behavior for regulation of ES cells proliferation by this FN isoform and could contribute to improve their culturing conditions both for research and cell therapy. PMID:24244705

  1. Mechanisms Regulating Stemness and Differentiation in Embryonal Carcinoma Cells

    Directory of Open Access Journals (Sweden)

    Gregory M. Kelly

    2017-01-01

    Full Text Available Just over ten years have passed since the seminal Takahashi-Yamanaka paper, and while most attention nowadays is on induced, embryonic, and cancer stem cells, much of the pioneering work arose from studies with embryonal carcinoma cells (ECCs derived from teratocarcinomas. This original work was broad in scope, but eventually led the way for us to focus on the components involved in the gene regulation of stemness and differentiation. As the name implies, ECCs are malignant in nature, yet maintain the ability to differentiate into the 3 germ layers and extraembryonic tissues, as well as behave normally when reintroduced into a healthy blastocyst. Retinoic acid signaling has been thoroughly interrogated in ECCs, especially in the F9 and P19 murine cell models, and while we have touched on this aspect, this review purposely highlights how some key transcription factors regulate pluripotency and cell stemness prior to this signaling. Another major focus is on the epigenetic regulation of ECCs and stem cells, and, towards that end, this review closes on what we see as a new frontier in combating aging and human disease, namely, how cellular metabolism shapes the epigenetic landscape and hence the pluripotency of all stem cells.

  2. Embryonic stem cell biology: insights from molecular imaging.

    Science.gov (United States)

    Sallam, Karim; Wu, Joseph C

    2010-01-01

    Embryonic stem (ES) cells have therapeutic potential in disorders of cellular loss such as myocardial infarction, type I diabetes and neurodegenerative disorders. ES cell biology in living subjects was largely poorly understood until incorporation of molecular imaging into the field. Reporter gene imaging works by integrating a reporter gene into ES cells and using a reporter probe to induce a signal detectable by normal imaging modalities. Reporter gene imaging allows for longitudinal tracking of ES cells within the same host for a prolonged period of time. This has advantages over postmortem immunohistochemistry and traditional imaging modalities. The advantages include expression of reporter gene is limited to viable cells, expression is conserved between generations of dividing cells, and expression can be linked to a specific population of cells. These advantages were especially useful in studying a dynamic cell population such as ES cells and proved useful in elucidating the biology of ES cells. Reporter gene imaging identified poor integration of differentiated ES cells transplanted into host tissue as well as delayed donor cell death as reasons for poor long-term survival in vivo. This imaging technology also confirmed that ES cells indeed have immunogenic properties that factor into cell survival and differentiation. Finally, reporter gene imaging improved our understanding of the neoplastic risk of undifferentiated ES cells in forming teratomas. Despite such advances, much remains to be understood about ES cell biology to translate this technology to the bedside, and reporter gene imaging will certainly play a key role in formulating this understanding.

  3. A Novel Growth-Promoting Pathway Formed by GDNF-Overexpressing Schwann Cells Promotes Propriospinal Axonal Regeneration, Synapse formation, and Partial Recovery of Function after Spinal Cord Injury

    Science.gov (United States)

    Deng, Lingxiao; Deng, Ping; Ruan, Yiwen; Xu, Zao Cheng; Liu, Naikui; Wen, Xuejun; Smith, George M.; Xu, Xiao-Ming

    2013-01-01

    Descending propriospinal neurons (DPSN) are known to establish functional relays for supraspinal signals, and they display a greater growth response after injury than do the long projecting axons. However, their regenerative response is still deficient due to their failure to depart from growth supportive cellular transplants back into the host spinal cord, which contains numerous impediments to axon growth. Here we report the construction of a continuous growth-promoting pathway in adult rats, formed by grafted Schwann cells (SCs) overexpressing glial cell line-derived neurotrophic factor (GDNF). We demonstrate that such a growth-promoting pathway, extending from the axonal cut ends to the site of innervation in the distal spinal cord, promoted regeneration of DPSN axons through and beyond the lesion gap of a spinal cord hemisection. Within the distal host spinal cord, regenerated DPSN axons formed synapses with host neurons leading to the restoration of action potentials and partial recovery of function. PMID:23536080

  4. A novel growth-promoting pathway formed by GDNF-overexpressing Schwann cells promotes propriospinal axonal regeneration, synapse formation, and partial recovery of function after spinal cord injury.

    Science.gov (United States)

    Deng, Ling-Xiao; Deng, Ping; Ruan, Yiwen; Xu, Zao Cheng; Liu, Nai-Kui; Wen, Xuejun; Smith, George M; Xu, Xiao-Ming

    2013-03-27

    Descending propriospinal neurons (DPSN) are known to establish functional relays for supraspinal signals, and they display a greater growth response after injury than do the long projecting axons. However, their regenerative response is still deficient due to their failure to depart from growth supportive cellular transplants back into the host spinal cord, which contains numerous impediments to axon growth. Here we report the construction of a continuous growth-promoting pathway in adult rats, formed by grafted Schwann cells overexpressing glial cell line-derived neurotrophic factor (GDNF). We demonstrate that such a growth-promoting pathway, extending from the axonal cut ends to the site of innervation in the distal spinal cord, promoted regeneration of DPSN axons through and beyond the lesion gap of a spinal cord hemisection. Within the distal host spinal cord, regenerated DPSN axons formed synapses with host neurons leading to the restoration of action potentials and partial recovery of function.

  5. Somatic donor cell type correlates with embryonic, but not extra-embryonic, gene expression in postimplantation cloned embryos.

    Directory of Open Access Journals (Sweden)

    Ryutaro Hirasawa

    Full Text Available The great majority of embryos generated by somatic cell nuclear transfer (SCNT display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts. The embryos retrieved from the uteri were separated into embryonic (epiblast and extraembryonic (extraembryonic ectoderm and ectoplacental cone tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by in vitro fertilization were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs (>2-fold vs. controls than did the extraembryonic tissues (P<1.0 × 10(-26. In the embryonic tissues, one of the common abnormalities was upregulation of Dlk1, a paternally imprinted gene. This might be a potential cause of the occasional placenta-only conceptuses seen in SCNT-generated mouse embryos (1-5% per embryos transferred in our laboratory, because dysregulation of the same gene is known to cause developmental failure of embryos derived from induced pluripotent stem cells. There were also some DEGs in the extraembryonic tissues, which might explain the poor development of SCNT-derived placentas at early stages. These findings suggest that SCNT affects the embryonic and extraembryonic development differentially and might cause further deterioration in the embryonic lineage in a donor cell-specific manner. This could explain donor cell-dependent variations in cloning efficiency using SCNT.

  6. Enhanced expression of FNDC5 in human embryonic stem cell-derived neural cells along with relevant embryonic neural tissues.

    Science.gov (United States)

    Ghahrizjani, Fatemeh Ahmadi; Ghaedi, Kamran; Salamian, Ahmad; Tanhaei, Somayeh; Nejati, Alireza Shoaraye; Salehi, Hossein; Nabiuni, Mohammad; Baharvand, Hossein; Nasr-Esfahani, Mohammad Hossein

    2015-02-25

    Availability of human embryonic stem cells (hESCs) has enhanced the capability of basic and clinical research in the context of human neural differentiation. Derivation of neural progenitor (NP) cells from hESCs facilitates the process of human embryonic development through the generation of neuronal subtypes. We have recently indicated that fibronectin type III domain containing 5 protein (FNDC5) expression is required for appropriate neural differentiation of mouse embryonic stem cells (mESCs). Bioinformatics analyses have shown the presence of three isoforms for human FNDC5 mRNA. To differentiate which isoform of FNDC5 is involved in the process of human neural differentiation, we have used hESCs as an in vitro model for neural differentiation by retinoic acid (RA) induction. The hESC line, Royan H5, was differentiated into a neural lineage in defined adherent culture treated by RA and basic fibroblast growth factor (bFGF). We collected all cell types that included hESCs, rosette structures, and neural cells in an attempt to assess the expression of FNDC5 isoforms. There was a contiguous increase in all three FNDC5 isoforms during the neural differentiation process. Furthermore, the highest level of expression of the isoforms was significantly observed in neural cells compared to hESCs and the rosette structures known as neural precursor cells (NPCs). High expression levels of FNDC5 in human fetal brain and spinal cord tissues have suggested the involvement of this gene in neural tube development. Additional research is necessary to determine the major function of FDNC5 in this process. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Co- transplantation of Bone Marrow Stromal Cells with Schwann Cells Evokes Mechanical Allodynia in the Contusion Model of Spinal Cord Injury in Rats

    Science.gov (United States)

    Pourheydar, Bagher; Joghataei, Mohammad Taghi; Bakhtiari, Mehrdad; Mehdizadeh, Mehdi; Yekta, Zahra; Najafzadeh, Norooz

    2012-01-01

    Objective: Several studies have shown that, although transplantation of neural stem cells into the contusion model of spinal cord injury (SCI) promotes locomotor function and improves functional recovery, it induces a painful response, Allodynia. Different studies indicate that bone marrow stromal cells (BMSCs) and Schwann cells (SCs) can improve locomotor recovery when transplanted into the injured rat spinal cord. Since these cells are commonly used in cell therapy, we investigated whether co-transplantation of these cells leads to the development of Allodynia. Materials and Methods: In this experimental research, the contusion model of SCI was induced by laminectomy at the T8-T9 level of the spinal cord in adult female wistar rats (n=40) weighting (250-300g) using the New York University Device. BMSCs and SCs were cultured and prelabeled with 5-bromo-2-deoxyuridine (BrdU) and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) respectively. The rats were divided into five groups of 8 including: a control group (laminectomy only), three experimental groups (BMSC, SC and Co-transplant) and a sham group. The experimental groups received BMSCs, SCs, and BMSCs and SCs respectively by intraspinal injection 7 days after injury and the sham group received serum only. Locomotion was assessed using Basso, Beattie and Bresnahan (BBB) test and Allodynia by the withdrawal threshold test using Von Frey Filaments at 1, 7, 14, 21, 28, 35, 42, 49 and 56 days after SCI. The statistical comparisons between groups were carried out by using repeated measures analysis of variances (ANOVA). Results: Significant differences were observed in BBB scores in the Co- transplant group compared to the BMSC and SC groups (pspinal cord can improve functional recovery, it leads to the development of mechanical Allodynia. This finding indicates that strategies to reduce Allodynia in cell transplantation studies are required. PMID:23508042

  8. Schwann cell-specific JAM-C-deficient mice reveal novel expression and functions for JAM-C in peripheral nerves

    Science.gov (United States)

    Colom, Bartomeu; Poitelon, Yannick; Huang, Wenlong; Woodfin, Abigail; Averill, Sharon; Del Carro, Ubaldo; Zambroni, Desirée; Brain, Susan D.; Perretti, Mauro; Ahluwalia, Amrita; Priestley, John V.; Chavakis, Triantafyllos; Imhof, Beat A.; Feltri, M. Laura; Nourshargh, Sussan

    2012-01-01

    Junctional adhesion molecule-C (JAM-C) is an adhesion molecule expressed at junctions between adjacent endothelial and epithelial cells and implicated in multiple inflammatory and vascular responses. In addition, we recently reported on the expression of JAM-C in Schwann cells (SCs) and its importance for the integrity and function of peripheral nerves. To investigate the role of JAM-C in neuronal functions further, mice with a specific deletion of JAM-C in SCs (JAM-C SC KO) were generated. Compared to wild-type (WT) controls, JAM-C SC KO mice showed electrophysiological defects, muscular weakness, and hypersensitivity to mechanical stimuli. In addressing the underlying cause of these defects, nerves from JAM-C SC KO mice were found to have morphological defects in the paranodal region, exhibiting increased nodal length as compared to WTs. The study also reports on previously undetected expressions of JAM-C, namely on perineural cells, and in line with nociception defects of the JAM-C SC KO animals, on finely myelinated sensory nerve fibers. Collectively, the generation and characterization of JAM-C SC KO mice has provided unequivocal evidence for the involvement of SC JAM-C in the fine organization of peripheral nerves and in modulating multiple neuronal responses.—Colom, B., Poitelon, Y., Huang, W., Woodfin, A., Averill, S., Del Carro, U., Zambroni, D., Brain, S. D., Perretti, M., Ahluwalia, A., Priestley, J. V., Chavakis, T., Imhof, B. A., Feltri, M. L., Nourshargh, S. Schwann cell-specific JAM-C-deficient mice reveal novel expression and functions for JAM-C in peripheral nerves. PMID:22090315

  9. The ethical dilemma of embryonic stem cell research.

    Science.gov (United States)

    Manzar, Nabeel; Manzar, Bushra; Hussain, Nuzhat; Hussain, M Fawwad Ahmed; Raza, Sajjad

    2013-03-01

    To determine the knowledge, attitude, and ethical concerns of medical students and graduates with regard to Embryonic Stem Cell (ESC) research. This questionnaire based descriptive study was conducted at the Civil Hospital Karachi (CHK), Pakistan from February to July 2008. A well structured questionnaire was administered to medical students and graduate doctors, which included their demographic profile as well as questions in line with the study objective. Informed consent was taken and full confidentiality was assured to the participants. Data were entered in a Statistical Package for Social Sciences (SPSS version.12) and analyzed. A total of 204 male and 216 female medical students and doctors were administered questionnaires out of which 105 males (51.4%) and 108 females (50%) were aware of the embryonic stem cell research and its ethical implications. Forty percent males and 47% of females were of the opinion that life begins at conception. Forty-six percent males and 39% females were in favor of stem cell research while only 31% males and 28% females supported the ESC research. Less than 1/3 of students supported using frozen embryos for research purposes while more than 2/3 indicated that they were unlikely to support abortion for stem cell research purposes. The majority of the students were in favor of stem cell research with some reservations regarding ESC research. A sizeable number of students withheld their views, reflecting their poor understanding of medical ethics. The result of the study indicates a need for incorporating bioethics into the medical curriculum.

  10. Embryonic Stem Cell Proteins and MicroRNAs in the Etiology of Germ Cell Cancer

    NARCIS (Netherlands)

    R. Eini (Ronak)

    2013-01-01

    textabstractIn the early 1980s, a population of unique cells was isolated from the inner cell mass (ICM) of the mouse pre-implantation embryo named embryonic stem (ES) cells. These cells were generated by removing the ICM from pre-implantation blastocysts. The resulting cells were found to be

  11. Derivation and characterization of a pig embryonic stem cell-derived exocrine pancreatic cell line

    Science.gov (United States)

    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...

  12. A Focused Microarray for Screening Rat Embryonic Stem Cell Lines

    Science.gov (United States)

    Hong, James; He, Hong; Bui, Phuoc; Ryba-White, Ben; Rumi, Mohammad A.K.; Soares, Michael J.; Dutta, Debasree; Paul, Soumen; Kawamata, Masaki; Ochiya, Takahiro; Ying, Qi-Long; Rajanahalli, Pavan

    2013-01-01

    Here, we describe a focused microarray for screening rat embryonic stem cells (ESCs) and provide validation data that this array can distinguish undifferentiated rat ESCs from rat trophoblast stem (TS) cells, rat extraembryonic endoderm cells, mouse embryonic fibroblast feeder cells, and differentiated rat ESCs. Using this tool, genuine rat ESC lines, which have been expanded in a conventional rat ESC medium containing two inhibitors (2i), for example, glycogen synthase kinase 3 (GSK3) and mitogen-activated protein kinase (MEK) inhibitors, and leukemia inhibitory factor, and genuine rat ESCs, which have been expanded in rat ESC medium containing four inhibitors (4i), for example, GSK3, MEK, Alk5, and Rho-associated kinase inhibitors were compared; as were genuine rat ESCs from 4 different strains of rats. Expression of Cdx2, a gene associated with trophoblast determination, was observed in genuine, undifferentiated rat ESCs from 4 strains and from both 2i and 4i ESC derivation medium. This finding is in contrast to undifferentiated mouse ESCs that do not express Cdx2. The rat ESC focused microarray described in this report has utility for rapid screening of rat ESCs. This tool will enable optimization of culture conditions in the future. PMID:22889370

  13. Tracking the mechanical dynamics of human embryonic stem cell chromatin

    Directory of Open Access Journals (Sweden)

    Hinde Elizabeth

    2012-12-01

    Full Text Available Abstract Background A plastic chromatin structure has emerged as fundamental to the self-renewal and pluripotent capacity of embryonic stem (ES cells. Direct measurement of chromatin dynamics in vivo is, however, challenging as high spatiotemporal resolution is required. Here, we present a new tracking-based method which can detect high frequency chromatin movement and quantify the mechanical dynamics of chromatin in live cells. Results We use this method to study how the mechanical properties of chromatin movement in human embryonic stem cells (hESCs are modulated spatiotemporally during differentiation into cardiomyocytes (CM. Notably, we find that pluripotency is associated with a highly discrete, energy-dependent frequency of chromatin movement that we refer to as a ‘breathing’ state. We find that this ‘breathing’ state is strictly dependent on the metabolic state of the cell and is progressively silenced during differentiation. Conclusions We thus propose that the measured chromatin high frequency movements in hESCs may represent a hallmark of pluripotency and serve as a mechanism to maintain the genome in a transcriptionally accessible state. This is a result that could not have been observed without the high spatial and temporal resolution provided by this novel tracking method.

  14. Apoptosis during embryonic tissue remodeling is accompanied by cell senescence

    Science.gov (United States)

    Lorda-Diez, Carlos I.; Garcia-Riart, Beatriz; Montero, Juan A.; Rodriguez-León, Joaquín; Garcia-Porrero, Juan A; Hurle, Juan M.

    2015-01-01

    This study re-examined the dying process in the interdigital tissue during the formation of free digits in the developing limbs. We demonstrated that the interdigital dying process was associated with cell senescence, as deduced by induction of β-gal activity, mitotic arrest, and transcriptional up-regulation of p21 together with many components of the senescence-associated secretory phenotype. We also found overlapping domains of expression of members of the Btg/Tob gene family of antiproliferative factors in the regressing interdigits. Notably, Btg2 was up-regulated during interdigit remodeling in species with free digits but not in the webbed foot of the duck. We also demonstrate that oxidative stress promoted the expression of Btg2, and that FGF2 and IGF1 which are survival signals for embryonic limb mesenchyme inhibited Btg2 expression. Btg2 overexpression in vivo and in vitro induced all the observed changes during interdigit regression, including oxidative stress, arrest of cell cycle progression, transcriptional regulation of senescence markers, and caspase-mediated apoptosis. Consistent with the central role of p21 on cell senescence, the transcriptional effects induced by overexpression of Btg2 are attenuated by silencing p21. Our findings indicate that cell senescence and apoptosis are complementary processes in the regression of embryonic tissues and share common regulatory signals. PMID:26568417

  15. Schwann cells and mesenchymal stem cells in laminin- or fibronectin-aligned matrices and regeneration across a critical size defect of 15 mm in the rat sciatic nerve.

    Science.gov (United States)

    Gonzalez-Perez, Francisco; Hernández, Joaquim; Heimann, Claudia; Phillips, James B; Udina, Esther; Navarro, Xavier

    2018-01-01

    OBJECTIVE Artificial nerve guides are being developed to substitute for autograft repair after peripheral nerve injuries. However, the use of conduits is limited by the length of the gap that needs to be bridged, with the success of regeneration highly compromised in long gaps. Addition of aligned proregenerative cells and extracellular matrix (ECM) components inside the conduit can be a good strategy to achieve artificial grafts that recreate the natural environment offered by a nerve graft. The purpose of this study was to functionalize chitosan devices with different cell types to support regeneration in limiting gaps in the rat peripheral nerve. METHODS The authors used chitosan devices combined with proteins of the ECM and cells in a rat model of sciatic nerve injury. Combinations of fibronectin and laminin with mesenchymal stem cells (MSCs) or Schwann cells (SCs) were aligned within tethered collagen-based gels, which were placed inside chitosan tubes that were then used to repair a critical-size gap of 15 mm in the rat sciatic nerve. Electrophysiology and algesimetry tests were performed to analyze functional recovery during the 4 months after injury and repair. Histological analysis was performed at the midlevel and distal level of the tubes to assess the number of regenerated myelinated fibers. RESULTS Functional analysis demonstrated that SC-aligned scaffolds resulted in 100% regeneration success in a 15-mm nerve defect in this rat model. In contrast, animals that underwent repair with MSC-aligned constructs had only 90% regeneration success, and those implanted with acellular bridges had only 75% regeneration success. CONCLUSIONS These results indicate that the combination of chitosan conduits with ECM-enriched cellular gels represents a good alternative to the use of autografts for repairing long nerve gaps.

  16. A Sweet Potion to Put Embryonic Stem Cells to Sleep

    Directory of Open Access Journals (Sweden)

    Kaushik D. Deb

    2009-01-01

    Full Text Available Human embryonic stem cells (hESCs are rapidly revolutionizing the areas of drug screening and therapy. In view of their applications and high operational costs at global multicentric setups, the ability to store and transport hESCs and derivatives under ambient temperatures, and their cryopreservation without compromising the stemness, function, and viability, is becoming imperative. Here we discuss the need for a natural cryoprotectant and biopreservative with a potential to improve cryopreservation, ambient temperature storage, and shipping of hESCs and derivatives. Trehalose, a naturally occurring disaccharide with therapeutic properties, protects the integrity of cells against desiccation, dehydration, and extreme heat or cold, and has been successfully tested for some somatic stem cell types. However, the biggest setback is the inability of mammalian cells to internalize trehalose. Here we review the methods being developed at different laboratories to facilitate its intercellular transport and advocate the need for similar advances in hESCs.

  17. Measurement of human embryonic stem cell in the growing cycle

    Science.gov (United States)

    Li, X.; Zhao, L.; Oh, Steve K. W.; Chong, W. K.; Ong, J. K.; Chen, Allen K.; Choo, Andre B. H.

    2008-09-01

    A measurement and imaging system has been developed for in-line continuous measurement of live, unmodified, human embryonic stem cells (hESC). The measurement will not affect cell growth, structure, sterility and suitability for clinical use. The stem cell imaging system (SCIS) can be used to support the optimization of automated stem cell growth for invitro study and for high-volume bio-manufacture. This paper present the experimental and analysis for the optimization of system parameters. A non-linear lighting is developed to obtain a clear images. The individual cluster can be traced from day one to day two. The whole system is calibrated with measurement microscope and haemacytometer. The measurement accuracy is better than 90%.

  18. Chromatin regulation landscape of embryonic stem cell identity.

    Science.gov (United States)

    Lee, Yun Hwa; Wu, Qiang

    2011-04-01

    ES cells (embryonic stem cells) derived from the ICM (inner cell mass) of blastocysts are pluripotent and are capable of giving rise to most cell types. The ES cell identity is mainly maintained by the Oct4 (octamer-binding transcription factor 4) and Nanog transcriptional networks. Recently, a tremendous amount of work has focused on deciphering how ES cell identity is regulated epigenetically. It has been shown that histone methylation/demethylation, histone acetylation/deacetylation, histone variants and chromatin remodelling play crucial roles in ES cell maintenance and differentiation. Moreover, perturbation of those chromatin regulators results in loss of ES cell identity or aberrant differentiation. Therefore, it is important to fully understand the chromatin regulation landscape of ES cells. The knowledge gained will help us to harness the unique characteristics of ES cells for stem cell-related therapy and regenerative medicine. In the present review, we will discuss recent proceedings that provide novel insights into chromatin regulation of ES cell identity.

  19. Synchronous Quadruple Primary Neoplasms: Colon Adenocarcinoma, Collision Tumor of Neuroendocrine Tumor and Schwann Cell Hamartoma and Sessile Serrated Adenoma of the Appendix.

    Science.gov (United States)

    Meeks, Marshall W; Grace, Shane; Chen, Yongxin; Petterchak, James; Bolesta, Edward; Zhou, Yihua; Lai, Jin-Ping

    2016-08-01

    Quadruple synchronous primary neoplasms are very rare with only three cases reported in the English-speaking literature to date. Collision tumors are also rare entities, especially of the appendix. We herein report a case of synchronous quadruple primary neoplasm in a 95-year-old female. She was diagnosed with colon adenocarcinoma, sessile serrated adenoma of the appendix and a collision tumor composed of a well-differentiated neuroendocrine tumor and Schwann cell hamartoma. Histological examination and immunohistochemistry supported these four lesions as separate entities. This case is unique because we report the diagnosis of quadruple synchronous primary, an extremely rare occurrence, in addition to a collision tumor of the appendix. We also provide a review of the literature for synchronous neoplasms and collision tumors. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  20. Tyrosine phosphatases epsilon and alpha perform specific and overlapping functions in regulation of voltage-gated potassium channels in Schwann cells

    DEFF Research Database (Denmark)

    Tiran, Zohar; Peretz, Asher; Sines, Tal

    2006-01-01

    + channels and Src were analyzed in vivo in mice lacking either or both PTPs. Lack of either PTP increases Kv channel activity and phosphorylation in Schwann cells, indicating these PTPs inhibit Kv current amplitude in vivo. Open probability and unitary conductance of Kv channels are unchanged, suggesting......Tyrosine phosphatases (PTPs) epsilon and alpha are closely related and share several molecular functions, such as regulation of Src family kinases and voltage-gated potassium (Kv) channels. Functional interrelationships between PTPepsilon and PTPalpha and the mechanisms by which they regulate K...... an effect on channel number or organization. PTPalpha inhibits Kv channels more strongly than PTPepsilon; this correlates with constitutive association of PTPalpha with Kv2.1, driven by membranal localization of PTPalpha. PTPalpha, but not PTPepsilon, activates Src in sciatic nerve extracts, suggesting Src...

  1. Tang-Luo-Ning, a Traditional Chinese Medicine, Inhibits Endoplasmic Reticulum Stress-Induced Apoptosis of Schwann Cells under High Glucose Environment

    Directory of Open Access Journals (Sweden)

    Weijie Yao

    2017-01-01

    Full Text Available Tang-Luo-Ning (TLN has a definite effect in the clinical treatment of diabetic peripheral neuropathy (DPN. Schwann cells (SCs apoptosis induced by endoplasmic reticulum stress (ER stress is one of the main pathogeneses of DPN. This study investigates whether TLN can inhibit SCs apoptosis by inhibiting ER stress-induced apoptosis. Our previous researches have demonstrated that TLN could increase the expression of ER stress marker protein GRP78 and inhibited the expression of apoptosis marker protein CHOP in ER stress. In this study, the results showed that TLN attenuated apoptosis by decreasing Ca2+ level in SCs and maintaining ER morphology. TLN could decrease downstream proteins of CHOP including GADD34 and Ero1α, while it increased P-eIF2α and decreased the upstream proteins of CHOP including P-IRE1α/IRE1α and XBP-1, thereby reducing ER stress-induced apoptosis.

  2. An animal model of Miller Fisher syndrome: Mitochondrial hydrogen peroxide is produced by the autoimmune attack of nerve terminals and activates Schwann cells.

    Science.gov (United States)

    Rodella, Umberto; Scorzeto, Michele; Duregotti, Elisa; Negro, Samuele; Dickinson, Bryan C; Chang, Christopher J; Yuki, Nobuhiro; Rigoni, Michela; Montecucco, Cesare

    2016-12-01

    The neuromuscular junction is a tripartite synapse composed of the presynaptic nerve terminal, the muscle and perisynaptic Schwann cells. Its functionality is essential for the execution of body movements and is compromised in a number of disorders, including Miller Fisher syndrome, a variant of Guillain-Barré syndrome: this autoimmune peripheral neuropathy is triggered by autoantibodies specific for the polysialogangliosides GQ1b and GT1a present in motor axon terminals, including those innervating ocular muscles, and in sensory neurons. Their binding to the presynaptic membrane activates the complement cascade, leading to a nerve degeneration that resembles that caused by some animal presynaptic neurotoxins. Here we have studied the intra- and inter-cellular signaling triggered by the binding and complement activation of a mouse monoclonal anti-GQ1b/GT1a antibody to primary cultures of spinal cord motor neurons and cerebellar granular neurons. We found that a membrane attack complex is rapidly assembled following antibody binding, leading to calcium accumulation, which affects mitochondrial functionality. Consequently, using fluorescent probes specific for mitochondrial hydrogen peroxide, we found that this reactive oxygen species is rapidly produced by mitochondria of damaged neurons, and that it triggers the activation of the MAP kinase pathway in Schwann cells. These results throw light on the molecular and cellular pathogenesis of Miller Fisher syndrome, and may well be relevant to other pathologies of the motor axon terminals, including some subtypes of the Guillain Barré syndrome. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  3. Transcriptional profiling of Sox17-positive, endoderm-committed mouse embryonic stem cells

    OpenAIRE

    Nolden, Tobias

    2010-01-01

    The major first step in embryonic development of vertebrates is the formation of three germ layers during the process of gastrulation - the ectoderm, the mesoderm and the endoderm. During gastrulation, embryonic cells of the epiblast loose their pluripotency and adopt different developmental fates. However, the underlying molecular mechanisms have not been fully elucidated. Embryonic stem cells (ESCs) derived from the epiblast largely express the same genes as the pluripotent epiblast cells ...

  4. Early gene regulation of osteogenesis in embryonic stem cells

    KAUST Repository

    Kirkham, Glen R.

    2012-01-01

    The early gene regulatory networks (GRNs) that mediate stem cell differentiation are complex, and the underlying regulatory associations can be difficult to map accurately. In this study, the expression profiles of the genes Dlx5, Msx2 and Runx2 in mouse embryonic stem cells were monitored over a 48 hour period after exposure to the growth factors BMP2 and TGFβ1. Candidate GRNs of early osteogenesis were constructed based on published experimental findings and simulation results of Boolean and ordinary differential equation models were compared with our experimental data in order to test the validity of these models. Three gene regulatory networks were found to be consistent with the data, one of these networks exhibited sustained oscillation, a behaviour which is consistent with the general view of embryonic stem cell plasticity. The work cycle presented in this paper illustrates how mathematical modelling can be used to elucidate from gene expression profiles GRNs that are consistent with experimental data. © 2012 The Royal Society of Chemistry.

  5. Species-specific control of cellular proliferation and the impact of large animal models for the use of olfactory ensheathing cells and Schwann cells in spinal cord repair.

    Science.gov (United States)

    Wewetzer, Konstantin; Radtke, Christine; Kocsis, Jeffery; Baumgärtner, Wolfgang

    2011-05-01

    Autologous transplantation of olfactory ensheathing cells (OECs) and Schwann cells (SCs) is considered a promising option to promote axonal regrowth and remyelination after spinal cord injury in humans. However, if the experimental data from the rodent model can be directly extrapolated to humans, as widely believed, remains to be established. While limitations of the rodent system have recently been discussed with regard to the distinct organization of the motor systems, the question whether OECs and SCs may display species-specific properties has not been fully addressed. Prompted by recent studies on canine and porcine glia, we performed a detailed analysis of the in vitro and in vivo properties of OECs and SCs and show that rodent but not human, monkey, porcine, and canine glia require mitogens for in vitro expansion, display a complex response to elevated intracellular cAMP, and undergo spontaneous immortalization upon prolonged mitogen stimulation. These data indicate fundamental inter-species differences of the control of cellular proliferation. Whether OECs and SCs from large animals and humans share growth-promoting in vivo properties with their rodent counterpart is not yet clear. Autologous implantation studies in humans did not reveal adverse effects of cell transplantation so far. However, in vivo studies of large animal or human glia and rodent recipients mainly focused on the remyelinating potential of the transplanted cells. Thus, further experimental in vivo studies in large animals are essential to fully define the axonal growth-promoting potential of OECs and SCs. Based on the homology of the in vitro growth control between porcine, canine and human glia, it is concluded that these species may serve as valuable translational models for scaling up human procedures. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair. Copyright © 2010 Elsevier Inc. All rights

  6. Embryonic stem cell research and the argument of complicity.

    Science.gov (United States)

    Birnbacher, Dieter

    2009-01-01

    While the argument of complicity is only rarely discussed in bioethics, it is of obvious relevance to the issue of imported embryonic stem cells in countries in which the derivation of stem cells from early human embryos is legally prohibited and/or morally rejected. Complicity means that making use of the results or products of an illegal or morally problematic activity is itself morally problematic, although generally to a lesser degree than the original activity. The question arises as to which conditions make the argument of complicity plausible, thus supporting attacks against legislation that aims to promote research based on 'fruits of a forbidden tree'. This paper distinguishes a number of different variants of complicity, proposes that they deserve different kinds of moral valuation and applies the results to the ongoing debate about the German stem cell research law.

  7. The N-glycome of human embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Olonen Anne

    2009-06-01

    Full Text Available Abstract Background Complex carbohydrate structures, glycans, are essential components of glycoproteins, glycolipids, and proteoglycans. While individual glycan structures including the SSEA and Tra antigens are already used to define undifferentiated human embryonic stem cells (hESC, the whole spectrum of stem cell glycans has remained unknown. We undertook a global study of the asparagine-linked glycoprotein glycans (N-glycans of hESC and their differentiated progeny using MALDI-TOF mass spectrometric and NMR spectroscopic profiling. Structural analyses were performed by specific glycosidase enzymes and mass spectrometric fragmentation analyses. Results The data demonstrated that hESC have a characteristic N-glycome which consists of both a constant part and a variable part that changes during hESC differentiation. hESC-associated N-glycans were downregulated and new structures emerged in the differentiated cells. Previously mouse embryonic stem cells have been associated with complex fucosylation by use of SSEA-1 antibody. In the present study we found that complex fucosylation was the most characteristic glycosylation feature also in undifferentiated hESC. The most abundant complex fucosylated structures were Lex and H type 2 antennae in sialylated complex-type N-glycans. Conclusion The N-glycan phenotype of hESC was shown to reflect their differentiation stage. During differentiation, hESC-associated N-glycan features were replaced by differentiated cell-associated structures. The results indicated that hESC differentiation stage can be determined by direct analysis of the N-glycan profile. These results provide the first overview of the N-glycan profile of hESC and form the basis for future strategies to target stem cell glycans.

  8. Embryoid body formation from embryonic and induced pluripotent stem cells:Benefits of bioreactors

    National Research Council Canada - National Science Library

    Sasitorn Rungarunlert Mongkol Techakumphu Melinda K Pirity Andras Dinnyes

    2009-01-01

    Embryonic stem(ES)cells have the ability to differ-entiate into all germ layers,holding great promise not only for a model of early embryonic development but also for a robust cell source for cell-replacement therapies...

  9. Undifferentiated Embryonic Cell Transcription Factor 1 Regulates ESC Chromatin Organization and Gene Expression

    NARCIS (Netherlands)

    Kooistra, Susanne M.; van den Boom, Vincent; Thummer, Rajkumar P.; Johannes, Frank; Wardenaar, Rene; Tesson, Bruno M.; Veenhoff, Liesbeth M.; Fusetti, Fabrizia; O'Neill, Laura P.; Turner, Bryan M.; de Haan, Gerald; Eggen, Bart J. L.; O’Neill, Laura P.

    2010-01-01

    Previous reports showed that embryonic stem (ES) cells contain hyperdynamic and globally transcribed chromatin-properties that are important for ES cell pluripotency and differentiation. Here, we demonstrate a role for undifferentiated embryonic cell transcription factor 1 (UTF1) in regulating ES

  10. Gene function in early mouse embryonic stem cell differentiation

    Directory of Open Access Journals (Sweden)

    Campbell Pearl A

    2007-03-01

    Full Text Available Abstract Background Little is known about the genes that drive embryonic stem cell differentiation. However, such knowledge is necessary if we are to exploit the therapeutic potential of stem cells. To uncover the genetic determinants of mouse embryonic stem cell (mESC differentiation, we have generated and analyzed 11-point time-series of DNA microarray data for three biologically equivalent but genetically distinct mESC lines (R1, J1, and V6.5 undergoing undirected differentiation into embryoid bodies (EBs over a period of two weeks. Results We identified the initial 12 hour period as reflecting the early stages of mESC differentiation and studied probe sets showing consistent changes of gene expression in that period. Gene function analysis indicated significant up-regulation of genes related to regulation of transcription and mRNA splicing, and down-regulation of genes related to intracellular signaling. Phylogenetic analysis indicated that the genes showing the largest expression changes were more likely to have originated in metazoans. The probe sets with the most consistent gene changes in the three cell lines represented 24 down-regulated and 12 up-regulated genes, all with closely related human homologues. Whereas some of these genes are known to be involved in embryonic developmental processes (e.g. Klf4, Otx2, Smn1, Socs3, Tagln, Tdgf1, our analysis points to others (such as transcription factor Phf21a, extracellular matrix related Lama1 and Cyr61, or endoplasmic reticulum related Sc4mol and Scd2 that have not been previously related to mESC function. The majority of identified functions were related to transcriptional regulation, intracellular signaling, and cytoskeleton. Genes involved in other cellular functions important in ESC differentiation such as chromatin remodeling and transmembrane receptors were not observed in this set. Conclusion Our analysis profiles for the first time gene expression at a very early stage of m

  11. Electrophysiological properties of embryonic stem cell-derived neurons.

    Directory of Open Access Journals (Sweden)

    Jessica R Risner-Janiczek

    Full Text Available In vitro generation of functional neurons from embryonic stem (ES cells and induced pluripotent stem cells offers exciting opportunities for dissecting gene function, disease modelling, and therapeutic drug screening. To realize the potential of stem cells in these biomedical applications, a complete understanding of the cell models of interest is required. While rapid advances have been made in developing the technologies for directed induction of defined neuronal subtypes, most published works focus on the molecular characterization of the derived neural cultures. To characterize the functional properties of these neural cultures, we utilized an ES cell model that gave rise to neurons expressing the green fluorescent protein (GFP and conducted targeted whole-cell electrophysiological recordings from ES cell-derived neurons. Current-clamp recordings revealed that most neurons could fire single overshooting action potentials; in some cases multiple action potentials could be evoked by depolarization, or occurred spontaneously. Voltage-clamp recordings revealed that neurons exhibited neuronal-like currents, including an outward current typical of a delayed rectifier potassium conductance and a fast-activating, fast-inactivating inward current, typical of a sodium conductance. Taken together, these results indicate that ES cell-derived GFP(+ neurons in culture display functional neuronal properties even at early stages of differentiation.

  12. Dynamic heterogeneity and DNA methylation in embryonic stem cells.

    KAUST Repository

    Singer, Zakary S

    2014-07-01

    Cell populations can be strikingly heterogeneous, composed of multiple cellular states, each exhibiting stochastic noise in its gene expression. A major challenge is to disentangle these two types of variability and to understand the dynamic processes and mechanisms that control them. Embryonic stem cells (ESCs) provide an ideal model system to address this issue because they exhibit heterogeneous and dynamic expression of functionally important regulatory factors. We analyzed gene expression in individual ESCs using single-molecule RNA-FISH and quantitative time-lapse movies. These data discriminated stochastic switching between two coherent (correlated) gene expression states and burst-like transcriptional noise. We further showed that the "2i" signaling pathway inhibitors modulate both types of variation. Finally, we found that DNA methylation plays a key role in maintaining these metastable states. Together, these results show how ESC gene expression states and dynamics arise from a combination of intrinsic noise, coherent cellular states, and epigenetic regulation.

  13. Retroviral transcriptional regulation and embryonic stem cells: war and peace.

    Science.gov (United States)

    Schlesinger, Sharon; Goff, Stephen P

    2015-03-01

    Retroviruses have evolved complex transcriptional enhancers and promoters that allow their replication in a wide range of tissue and cell types. Embryonic stem (ES) cells, however, characteristically suppress transcription of proviruses formed after infection by exogenous retroviruses and also of most members of the vast array of endogenous retroviruses in the genome. These cells have unusual profiles of transcribed genes and are poised to make rapid changes in those profiles upon induction of differentiation. Many of the transcription factors in ES cells control both host and retroviral genes coordinately, such that retroviral expression patterns can serve as markers of ES cell pluripotency. This overlap is not coincidental; retrovirus-derived regulatory sequences are often used to control cellular genes important for pluripotency. These sequences specify the temporal control and perhaps "noisy" control of cellular genes that direct proper cell gene expression in primitive cells and their differentiating progeny. The evidence suggests that the viral elements have been domesticated for host needs, reflecting the wide-ranging exploitation of any and all available DNA sequences in assembling regulatory networks. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  14. Therapeutic approaches for treating hemophilia A using embryonic stem cells.

    Science.gov (United States)

    Kasuda, Shogo; Tatsumi, Kohei; Sakurai, Yoshihiko; Shima, Midori; Hatake, Katsuhiko

    2016-06-01

    Hemophilia A is an X-linked rescessive bleeding disorder that results from F8 gene aberrations. Previously, we established embryonic stem (ES) cells (tet-226aa/N6-Ainv18) that secrete human factor VIII (hFVIII) by introducing the human F8 gene in mouse Ainv18 ES cells. Here, we explored the potential of cell transplantation therapy for hemophilia A using the ES cells. Transplant tet-226aa/N6-Ainv18 ES cells were injected into the spleens of severe combined immunodeficiency (SCID) mice, carbon tetrachloride (CCl4)-pretreated wild-type mice, and CCl4-pretreated hemophilia A mice. F8 expression was induced by doxycycline in drinking water, and hFVIII-antigen production was assessed in all cell transplantation experiments. Injecting the ES cells into SCID mice resulted in an enhanced expression of the hFVIII antigen; however, teratoma generation was confirmed in the spleen. Transplantation of ES cells into wild-type mice after CCl4-induced liver injury facilitated survival and engraftment of transplanted cells without teratoma formation, resulting in hFVIII production in the plasma. Although CCl4 was lethal to most hemophilia A mice, therapeutic levels of FVIII activity, as well as the hFVIII antigen, were detected in surviving hemophilia A mice after cell transplantation. Immunolocalization results for hFVIII suggested that transplanted ES cells might be engrafted at the periportal area in the liver. Although the development of a safer induction method for liver regeneration is required, our results suggested the potential for developing an effective ES-cell transplantation therapeutic model for treating hemophilia A in the future. Copyright © 2016 King Faisal Specialist Hospital & Research Centre. Published by Elsevier Ltd. All rights reserved.

  15. Transcriptome coexpression map of human embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Mattson Mark P

    2006-05-01

    Full Text Available Abstract Background Human embryonic stem (ES cells hold great promise for medicine and science. The transcriptome of human ES cells has been studied in detail in recent years. However, no systematic analysis has yet addressed whether gene expression in human ES cells may be regulated in chromosomal domains, and no chromosomal domains of coexpression have been identified. Results We report the first transcriptome coexpression map of the human ES cell and the earliest stage of ES differentiation, the embryoid body (EB, for the analysis of how transcriptional regulation interacts with genomic structure during ES self-renewal and differentiation. We determined the gene expression profiles from multiple ES and EB samples and identified chromosomal domains showing coexpression of adjacent genes on the genome. The coexpression domains were not random, with significant enrichment in chromosomes 8, 11, 16, 17, 19, and Y in the ES state, and 6, 11, 17, 19 and 20 in the EB state. The domains were significantly associated with Giemsa-negative bands in EB, yet showed little correlation with known cytogenetic structures in ES cells. Different patterns of coexpression were revealed by comparative transcriptome mapping between ES and EB. Conclusion The findings and methods reported in this investigation advance our understanding of how genome organization affects gene expression in human ES cells and help to identify new mechanisms and pathways controlling ES self-renewal or differentiation.

  16. Sourcing human embryos for embryonic stem cell lines: Problems & perspectives

    Directory of Open Access Journals (Sweden)

    Rajvi H Mehta

    2014-01-01

    Full Text Available The ability to successfully derive human embryonic stem cells (hESC lines from human embryos following in vitro fertilization (IVF opened up a plethora of potential applications of this technique. These cell lines could have been successfully used to increase our understanding of human developmental biology, transplantation medicine and the emerging science of regenerative medicine. The main source for human embryos has been ′discarded′ or ′spare′ fresh or frozen human embryos following IVF. It is a common practice to stimulate the ovaries of women undergoing any of the assisted reproductive technologies (ART and retrieve multiple oocytes which subsequently lead to multiple embryos. Of these, only two or maximum of three embryos are transferred while the rest are cryopreserved as per the decision of the couple. In case a couple does not desire to ′cryopreserve′ their embryos then all the embryos remaining following embryo transfer can be considered ′spare′ or if a couple is no longer in need of the ′cryopreserved′ embryos then these also can be considered as ′spare′. But, the question raised by the ethicists is, "what about ′slightly′ over-stimulating a woman to get a few extra eggs and embryos? The decision becomes more difficult when it comes to ′discarded′ embryos. As of today, the quality of the embryos is primarily assessed based on morphology and the rate of development mainly judged by single point assessment. Despite many criteria described in the literature, the quality assessment is purely subjective. The question that arises is on the decision of ′discarding′ embryos. What would be the criteria for discarding embryos and the potential ′use′ of ESC derived from the ′abnormal appearing′ embryos? This paper discusses some of the newer methods to procure embryos for the derivation of embryonic stem cell lines which will respect the ethical concerns but still provide the source material.

  17. Human embryonic stem cells as models for aneuploid chromosomal syndromes.

    Science.gov (United States)

    Biancotti, Juan-Carlos; Narwani, Kavita; Buehler, Nicole; Mandefro, Berhan; Golan-Lev, Tamar; Yanuka, Ofra; Clark, Amander; Hill, David; Benvenisty, Nissim; Lavon, Neta

    2010-09-01

    Syndromes caused by chromosomal aneuploidies are widely recognized genetic disorders in humans and often lead to spontaneous miscarriage. Preimplantation genetic screening is used to detect chromosomal aneuploidies in early embryos. Our aim was to derive aneuploid human embryonic stem cell (hESC) lines that may serve as models for human syndromes caused by aneuploidies. We have established 25 hESC lines from blastocysts diagnosed as aneuploid on day 3 of their in vitro development. The hESC lines exhibited morphology and expressed markers typical of hESCs. They demonstrated long-term proliferation capacity and pluripotent differentiation. Karyotype analysis revealed that two-third of the cell lines carry a normal euploid karyotype, while one-third remained aneuploid throughout the derivation, resulting in eight hESC lines carrying either trisomy 13 (Patau syndrome), 16, 17, 21 (Down syndrome), X (Triple X syndrome), or monosomy X (Turner syndrome). On the basis of the level of single nucleotide polymorphism heterozygosity in the aneuploid chromosomes, we determined whether the aneuploidy originated from meiotic or mitotic chromosomal nondisjunction. Gene expression profiles of the trisomic cell lines suggested that all three chromosomes are actively transcribed. Our analysis allowed us to determine which tissues are most affected by the presence of a third copy of either chromosome 13, 16, 17 or 21 and highlighted the effects of trisomies on embryonic development. The results presented here suggest that aneuploid embryos can serve as an alternative source for either normal euploid or aneuploid hESC lines, which represent an invaluable tool to study developmental aspects of chromosomal abnormalities in humans.

  18. Acute Putrescine Supplementation with Schwann Cell Implantation Improves Sensory and Serotonergic Axon Growth and Functional Recovery in Spinal Cord Injured Rats.

    Science.gov (United States)

    Iorgulescu, J Bryan; Patel, Samik P; Louro, Jack; Andrade, Christian M; Sanchez, Andre R; Pearse, Damien D

    2015-01-01

    Schwann cell (SC) transplantation exhibits significant potential for spinal cord injury (SCI) repair and its use as a therapeutic modality has now progressed to clinical trials for subacute and chronic human SCI. Although SC implants provide a receptive environment for axonal regrowth and support functional recovery in a number of experimental SCI models, axonal regeneration is largely limited to local systems and the behavioral improvements are modest without additional combinatory approaches. In the current study we investigated whether the concurrent delivery of the polyamine putrescine, started either 30 min or 1 week after SCI, could enhance the efficacy of SCs when implanted subacutely (1 week after injury) into the contused rat spinal cord. Polyamines are ubiquitous organic cations that play an important role in the regulation of the cell cycle, cell division, cytoskeletal organization, and cell differentiation. We show that the combination of putrescine with SCs provides a significant increase in implant size, an enhancement in axonal (sensory and serotonergic) sparing and/or growth, and improved open field locomotion after SCI, as compared to SC implantation alone. These findings demonstrate that polyamine supplementation can augment the effectiveness of SCs when used as a therapeutic approach for subacute SCI repair.

  19. From embryonic stem cells to testicular germ cell cancer-- should we be concerned?

    DEFF Research Database (Denmark)

    Almstrup, Kristian; Sonne, Si Brask; Hoei-Hansen, Christina E

    2006-01-01

    that initial hypothesis but also indicating that CIS cells have a striking phenotypic similarity to embryonic stem cells (ESC). Many cancers have been proposed to originate from tissue-specific stem cells [so-called 'cancer stem cells' (CSC)] and we argue that CIS may be a very good example of a CSC......, but with exceptional features due to the retention of embryonic pluripotency. In addition, considering the fact that pre-invasive CIS cells are transformed from early fetal cells, possibly due to environmentally induced alterations of the niche, we discuss potential risks linked to the uncontrolled therapeutic use......Since the discovery of testicular carcinoma in situ (CIS) -- the precursor cell for the vast majority of germ cell tumours -- it has been proposed that CIS cells could be derived from transformed primordial germ cells or gonocytes. Here, we review recent discoveries not only substantiating...

  20. Construction of nerve guide conduits from cellulose/soy protein composite membranes combined with Schwann cells and pyrroloquinoline quinone for the repair of peripheral nerve defect

    Energy Technology Data Exchange (ETDEWEB)

    Luo, Lihua [Department of Biomedical Engineering, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China); Center of Molecular Medicine, School of Medicine, Hubei University of Arts and Sciences, Xiangyang 441053 (China); Gan, Li; Liu, Yongming; Tian, Weiqun; Tong, Zan [Department of Biomedical Engineering, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China); Wang, Xiong; Huselstein, Celine [Ingénierie Moléculaire et Physiopathologie Articulaire (IMoPA), UMR 7365 CNRS – Université de Lorraine, Biopôle, 54500 Vandoeuvre-lès-Nancy (France); Chen, Yun, E-mail: yunchen@whu.edu.cn [Department of Biomedical Engineering, School of Basic Medical Sciences, Wuhan University, Wuhan 430071 (China)

    2015-02-20

    Regeneration and functional reconstruction of peripheral nerve defects remained a significant clinical challenge. Nerve guide conduits, with seed cells or neurotrophic factors (NTFs), had been widely used to improve the repair and regeneration of injured peripheral nerve. Pyrroloquinoline quinone (PQQ) was an antioxidant that can stimulate nerve growth factors (NGFs) synthesis and accelerate the Schwann cells (SCs) proliferation and growth. In present study, three kinds of nerve guide conduits were constructed: one from cellulose/SPI hollow tube (CSC), another from CSC combined with SCs (CSSC), and the third one from CSSC combined with PQQ (CSSPC), respectively. And then they were applied to bridge and repair the sciatic nerve defect in rats, using autograft as control. Effects of different nerve guide conduits on the nerve regeneration were comparatively evaluated by general analysis, sciatic function index (SFI) and histological analysis (HE and TEM). Newly-formed regenerative nerve fibers were observed and running through the transparent nerve guide conduits 12 weeks after surgery. SFI results indicated that the reconstruction of motor function in CSSPC group was better than that in CSSC and CSC groups. HE images from the cross-sections and longitudinal-sections of the harvested regenerative nerve indicated that regenerative nerve fibers had been formed and accompanied with new blood vessels and matrix materials in the conduits. TEM images also showed that lots of fresh myelinated and non-myelinated nerve fibers had been formed. Parts of vacuolar, swollen and abnormal axons occurred in CSC and CSSC groups, while the vacuolization and swell of axons was the least serious in CSSPC group. These results indicated that CSSPC group had the most ability to repair and reconstruct the nerve structure and functions due to the comprehensive contributions from hollow CSC tube, SCs and PQQ. As a result, the CSSPC may have the potential for the applications as nerve guide

  1. Human embryonic stem cell registries: value, challenges and opportunities.

    Science.gov (United States)

    Luong, Mai X; Smith, Kelly P; Stein, Gary S

    2008-10-15

    The accelerating pace of human embryonic stem cell (hESC) research has created an urgent need for the development of hESC registries, information repositories intended to gather, organize and disseminate hESC information. Although of enormous value to this evolving field, registries face significant challenges to their development. These challenges include addressing the legal and ethical issues surrounding hESC derivation as well as complex intellectual property concerns. In addition to these issues, registries must develop tools to efficiently gather, validate and present many different types of hESC information from a variety of sources. Given the pace and regulatory complexities of this field, it is important that registries develop cooperative mechanisms to avoid duplication and more efficiently support hESC research. (c) 2008 Wiley-Liss, Inc.

  2. GATA-1 directly regulates Nanog in mouse embryonic stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Li, Wen-Zhong; Ai, Zhi-Ying [College of Life Sciences, Northwest A& F University, Yangling 712100 (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100 (China); Wang, Zhi-Wei [School of Life Sciences and Medical Center, University of Science and Technology of China, Hefei, Anhui 230027 (China); Chen, Lin-Lin [College of Life Sciences, Northwest A& F University, Yangling 712100 (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100 (China); Guo, Ze-Kun, E-mail: gzknwaf@126.com [College of Veterinary Medicine, Northwest A& F University, Yangling 712100 (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100 (China); Zhang, Yong, E-mail: zylabnwaf@126.com [College of Veterinary Medicine, Northwest A& F University, Yangling 712100 (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100 (China)

    2015-09-25

    Nanog safeguards pluripotency in mouse embryonic stem cells (mESCs). Insight into the regulation of Nanog is important for a better understanding of the molecular mechanisms that control pluripotency of mESCs. In a silico analysis, we identify four GATA-1 putative binding sites in Nanog proximal promoter. The Nanog promoter activity can be significantly repressed by ectopic expression of GATA-1 evidenced by a promoter reporter assay. Mutation studies reveal that one of the four putative binding sites counts for GATA-1 repressing Nanog promoter activity. Direct binding of GATA-1 on Nanog proximal promoter is confirmed by electrophoretic mobility shift assay and chromatin immunoprecipitation. Our data provide new insights into the expanded regulatory circuitry that coordinates Nanog expression. - Highlights: • The Nanog proximal promoter conceives functional element for GATA-1. • GATA-1 occupies the Nanog proximal promoter in vitro and in vivo. • GATA-1 transcriptionally suppresses Nanog.

  3. Radial glial cells defined and major intermediates between embryonic stem cells and CNS neurons.

    Science.gov (United States)

    Götz, Magdalena; Barde, Yves-Alain

    2005-05-05

    Radial glial cells have been identified as a major source of neurons during development. Here, we review the evidence for the distinct "glial" nature of radial glial cells and contrast these cells with their progenitors, the neuroepithelial cells. Recent results also suggest that not only during neurogenesis in vivo, but also during the differentiation of cultured embryonic stem cells toward neurons, progenitors with clear glial antigenic characteristics act as cellular intermediates.

  4. Llgl1 Connects Cell Polarity with Cell-Cell Adhesion in Embryonic Neural Stem Cells.

    Science.gov (United States)

    Jossin, Yves; Lee, Minhui; Klezovitch, Olga; Kon, Elif; Cossard, Alexia; Lien, Wen-Hui; Fernandez, Tania E; Cooper, Jonathan A; Vasioukhin, Valera

    2017-06-05

    Malformations of the cerebral cortex (MCCs) are devastating developmental disorders. We report here that mice with embryonic neural stem-cell-specific deletion of Llgl1 (Nestin-Cre/Llgl1fl/fl), a mammalian ortholog of the Drosophila cell polarity gene lgl, exhibit MCCs resembling severe periventricular heterotopia (PH). Immunohistochemical analyses and live cortical imaging of PH formation revealed that disruption of apical junctional complexes (AJCs) was responsible for PH in Nestin-Cre/Llgl1fl/fl brains. While it is well known that cell polarity proteins govern the formation of AJCs, the exact mechanisms remain unclear. We show that LLGL1 directly binds to and promotes internalization of N-cadherin, and N-cadherin/LLGL1 interaction is inhibited by atypical protein kinase C-mediated phosphorylation of LLGL1, restricting the accumulation of AJCs to the basolateral-apical boundary. Disruption of the N-cadherin-LLGL1 interaction during cortical development in vivo is sufficient for PH. These findings reveal a mechanism responsible for the physical and functional connection between cell polarity and cell-cell adhesion machineries in mammalian cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Brown adipogenesis of mouse embryonic stem cells in alginate microstrands

    Science.gov (United States)

    Unser, Andrea Mannarino

    The ability of brown adipocytes (fat cells) to dissipate energy as heat shows great promise for the treatment of obesity and other metabolic disorders. Employing pluripotent stem cells, with an emphasis on directed differentiation, may overcome many issues currently associated with primary fat cell cultures. However, brown adipocytes are difficult to transplant in vivo due to the instability of fat, in terms of necrosis and neovascularization, once injected. Thus, 3D cell culture systems that have the potential to mimic adipogenic microenvironments are needed, not only to advance brown fat implantation, but also to better understand the role of brown adipocytes in treating obesity. To address this need, we created 3D "Brown-Fat-in-Microstrands" by microfluidic synthesis of alginate hydrogel microstrands that encapsulated cells and directly induced cell differentiation into brown adipocytes, using mouse embryonic stem cells (ESCs) as a model of pluripotent stem cells and brown preadipocytes as a positive control. The effect of hydrogel formation parameters on brown adipogenesis was studied, leading to the establishment of "Brown-Fat-in-Microstrands". Brown adipocyte differentiation within microstrands was confirmed by lipid droplet accumulation, immunocytochemistry and qPCR analysis of gene expression of brown adipocyte marker uncoupling protein 1 (UCP1) in addition to adipocyte marker expression. Compared to a 2D approach, 3D differentiated "Brown-Fat-in-Microstrands" exhibited higher level of brown adipocyte marker expression. The functional analysis of "Brown-Fat-in-Microstrands" was attempted by measuring the mitochondrial activity of ESC-differentiated brown adipocytes in 3D using Seahorse XF24 3 Extracellular Flux Analyzer. The ability to create "Brown-Fat-in-Microstrands" from pluripotent stem cells opens up a new arena to understanding brown adipogenesis and its implications in obesity and metabolic disorders.

  6. Functional Role of Mst1/Mst2 in Embryonic Stem Cell Differentiation

    OpenAIRE

    Peng Li; Ying Chen; Kinglun Kingston Mak; Chun Kwok Wong; Chi Chiu Wang; Ping Yuan

    2013-01-01

    The Hippo pathway is an evolutionary conserved pathway that involves cell proliferation, differentiation, apoptosis and organ size regulation. Mst1 and Mst2 are central components of this pathway that are essential for embryonic development, though their role in controlling embryonic stem cells (ES cells) has yet to be exploited. To further understand the Mst1/Mst2 function in ES cell pluripotency and differentiation, we derived Mst1/Mst2 double knockout (Mst-/-) ES cells to completely pertur...

  7. Activity-induced Ca2+ signaling in perisynaptic Schwann cells of the early postnatal mouse is mediated by P2Y1 receptors and regulates muscle fatigue

    Science.gov (United States)

    Heredia, Dante J; Feng, Cheng-Yuan; Hennig, Grant W; Renden, Robert B

    2018-01-01

    Perisynaptic glial cells respond to neural activity by increasing cytosolic calcium, but the significance of this pathway is unclear. Terminal/perisynaptic Schwann cells (TPSCs) are a perisynaptic glial cell at the neuromuscular junction that respond to nerve-derived substances such as acetylcholine and purines. Here, we provide genetic evidence that activity-induced calcium accumulation in neonatal TPSCs is mediated exclusively by one subtype of metabotropic purinergic receptor. In P2ry1 mutant mice lacking these responses, postsynaptic, rather than presynaptic, function was altered in response to nerve stimulation. This impairment was correlated with a greater susceptibility to activity-induced muscle fatigue. Interestingly, fatigue in P2ry1 mutants was more greatly exacerbated by exposure to high potassium than in control mice. High potassium itself increased cytosolic levels of calcium in TPSCs, a response which was also reduced P2ry1 mutants. These results suggest that activity-induced calcium responses in TPSCs regulate postsynaptic function and muscle fatigue by regulating perisynaptic potassium. PMID:29384476

  8. Potential applications of keratinocytes derived from human embryonic stem cells.

    Science.gov (United States)

    Movahednia, Mohammad M; Kidwai, Fahad K; Jokhun, Doorgesh S; Squier, Christopher A; Toh, Wei Seong; Cao, Tong

    2016-01-01

    Although skin grafting is one of the most advanced cell therapy technique, wide application of skin substitutes is hampered by the difficulty in securing sufficient amount of epidermal substitute. Additionally, in understanding the progression of skin aging and disease, and in screening the cosmetic and pharmaceutical products, there is lack of a satisfactory human skin-specific in vitro model. Recently, human embryonic stem cells (hESCs) have been proposed as an unlimited and reliable cell source to obtain almost all cell types present in the human body. This review focuses on the potential off-the-shelf use of hESC-derived keratinocytes for future clinical applications as well as a powerful in vitro skin model to study skin function and integrity, host-pathogen interactions and disease pathogenesis. Furthermore, we discuss the industrial applications of hESC-derived keratinized multi-layer epithelium which provides a human-like test platform for understanding disease pathogenesis, evaluation of new therapeutic modalities and assessment of the safety and efficacy of skin cosmetics and therapeutics. Overall, we conclude that the hESC-derived keratinocytes have great potential for clinical, research and industrial applications. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Epigenetically reprogramming metastatic tumor cells with an embryonic microenvironment

    Science.gov (United States)

    Costa, Fabricio F; Seftor, Elisabeth A; Bischof, Jared M; Kirschmann, Dawn A; Strizzi, Luigi; Arndt, Kelly; de Fatima Bonaldo, Maria; Soares, Marcelo B; Hendrix, Mary JC

    2010-01-01

    We have previously shown that the microenvironment of human embryonic stem cells (hESCs) is able to change and reprogram aggressive cancer cells to a less aggressive state. Some mechanisms implicated in the phenotypic changes observed after this exposure are mainly associated with the Nodal signaling pathway, which plays a key role in tumor cell plasticity. However, several other molecular mechanisms might be related directly and/or indirectly to these changes, including microRNA (miRNA) regulation and DNA methylation. Aim: To further explore the epigenetic mechanisms potentially underlying the phenotypic changes that occur after exposing metastatic melanoma cells to a hESC microenvironment. Materials & Methods: A total of 365 miRNAs were screened using the TaqMan® Low Density Arrays. We also evaluated whether DNA methylation could be one of the factors regulating the expression of the inhibitor of Nodal, Lefty, in hESCs (where it is highly expressed) vs melanoma cells (where it is not expressed). Results: Using these experimental approaches, we identified miRNAs that are up- and down-regulated in melanoma cells exposed to a hESC microenvironment, such as miR-302a and miR-27b, respectively. We also demonstrate that Notch4 is one of the targets of miR-302a, which is upstream of Nodal. Additionally, one of the mechanisms that might explain the absence of the inhibitor of Nodal, Lefty, in cancer cells is silencing by DNA methylation, which provides new insights into the unregulated expression of Nodal in melanoma. Conclusion: These findings suggest that epigenetic changes such as DNA methylation and regulation by microRNAs might play a significant role in tumor cell plasticity and the metastatic phenotype. PMID:20495621

  10. Production of embryonic and fetal-like red blood cells from human induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Chan-Jung Chang

    Full Text Available We have previously shown that human embryonic stem cells can be differentiated into embryonic and fetal type of red blood cells that sequentially express three types of hemoglobins recapitulating early human erythropoiesis. We report here that we have produced iPS from three somatic cell types: adult skin fibroblasts as well as embryonic and fetal mesenchymal stem cells. We show that regardless of the age of the donor cells, the iPS produced are fully reprogrammed into a pluripotent state that is undistinguishable from that of hESCs by low and high-throughput expression and detailed analysis of globin expression patterns by HPLC. This suggests that reprogramming with the four original Yamanaka pluripotency factors leads to complete erasure of all functionally important epigenetic marks associated with erythroid differentiation regardless of the age or the tissue type of the donor cells, at least as detected in these assays. The ability to produce large number of erythroid cells with embryonic and fetal-like characteristics is likely to have many translational applications.

  11. Ginsenoside Re Promotes Nerve Regeneration by Facilitating the Proliferation, Differentiation and Migration of Schwann Cells via the ERK- and JNK-Dependent Pathway in Rat Model of Sciatic Nerve Crush Injury.

    Science.gov (United States)

    Wang, Lei; Yuan, Damin; Zhang, Dongmei; Zhang, Weidong; Liu, Chun; Cheng, Hongbing; Song, Yan; Tan, Qian

    2015-08-01

    Exploring effective drugs that are capable of promoting nerve regeneration has gained much attention. Ginsenoside Re (Re) is the main ingredient of ginseng berries and roots. Research in the area has shown that ginsenoside Re exhibits multiple pharmacological activities via different mechanisms both in vivo and in vitro. But the potential therapeutic effects of Re on sciatic nerve crush injury (SNC) have been little investigated. Herein, we investigated the protect effect of Re on peripheral nerve regeneration in a rat SNC model. Walking track analysis revealed that Re treatment significantly promoted functional recovery of crushed sciatic nerve in rats. The expression of PCNA in rat sciatic nerve was up-regulated by Re treatment, and peaked when the concentration of Re was 2.0 mg/kg. Using immunofluorescent staining, we found that Re greatly increased the expression of GAP-43 and S100 in injured rat sciatic nerve. Furthermore, we evaluated the effects of Re on proliferation, differentiation, and migration of Schwann cells in SNC rat models. Our studies reveal that Re promotes nerve regeneration is depend on ERK1/2 and JNK1/2 signaling pathway. Elevated Oct-6 expression and featured morphological changes indicated that Re facilitated the differentiation of Schwann cells following SNC. Also, transwell and wound-healing assay demonstrated that the migration capabilities of Schwann cell were significantly enhanced after Re treatment.

  12. Cotransplant of neural stem cells and NT-3 gene modified Schwann cells promote the recovery of transected spinal cord injury.

    Science.gov (United States)

    Guo, J-S; Zeng, Y-S; Li, H-B; Huang, W-L; Liu, R-Y; Li, X-B; Ding, Y; Wu, L-Z; Cai, D-Z

    2007-01-01

    An animal model of transected spinal cord injury (SCI) was used to test the hypothesis that cografted neural stem cells (NSCs) and NT-3-SCs promote morphologic and functional recoveries of injured spinal cord. To explore whether cotransplant of NSCs and NT-3-SCs could promote the injured spinal cord repair. Zhongshan Medical College, Sun Yat-sen University, PR China. Female Sprague-Dawley (SD) rats weighing on 200-220 g were used to prepare SCI models. The spinal cord was transected between T(9) and T(10), then NSCs, SCs+NSCs, LacZ-SCs+NSCs, or NT-3-SCs+NSCs were grafted into the transected site. (1) Part of NSCs could differentiate to neuron-like cells in the transected site and the percentage of differentiation was NT-3-SCs+NSCs group>SCs+NSCs group>NSCs group. (2) In the grafted groups, there were 5-HT, CGRP, and SP positive nerve fibres within the transected site. Some fluorogold (FG)-labeled cells were found in the spinal cord rostral to the transected site, the red nuclei and the inner pyramidal layer of sensorimotor cortex. (3) The cells grafted could enhance the injured neurons survival in inner pyramidal layer of sensorimotor cortex, red nuclei of midbrain, and Clark's nuclei of spinal cord's L1 segment, could decrease the latency and increase the amplitude of cortical somatosensory evoked potential (CSEP) and cortical motor evoked potential (CMEP), and could promote partly structural and functional recovery of the SCI rats. These results demonstrate that cografted NT-3-SCs and NSCs is a potential therapy for SCI. This research was supported by Chinese National Key Project for Basic Research (G1999054009), Chinese National Natural Science Foundation (30270700) and Social Developmental Foundation of Guangdong Province (2003C33808) to YS Zeng; Natural Science Foundation of Guangdong Province (04300468) and Medical Science Research Grant of Guangdong Province (A2004081) to JS Guo.

  13. Maternal Embryonic Leucine Zipper Kinase (MELK: A Novel Regulator in Cell Cycle Control, Embryonic Development, and Cancer

    Directory of Open Access Journals (Sweden)

    Pengfei Jiang

    2013-10-01

    Full Text Available Maternal embryonic leucine zipper kinase (MELK functions as a modulator of intracellular signaling and affects various cellular and biological processes, including cell cycle, cell proliferation, apoptosis, spliceosome assembly, gene expression, embryonic development, hematopoiesis, and oncogenesis. In these cellular processes, MELK functions by binding to numerous proteins. In general, the effects of multiple protein interactions with MELK are oncogenic in nature, and the overexpression of MELK in kinds of cancer provides some evidence that it may be involved in tumorigenic process. In this review, our current knowledge of MELK function and recent discoveries in MELK signaling pathway were discussed. The regulation of MELK in cancers and its potential as a therapeutic target were also described.

  14. Human embryonic stem cells have enhanced repair of multiple forms of DNA damage

    DEFF Research Database (Denmark)

    Maynard, Scott; Swistowska, Anna Maria; Lee, Jae Wan

    2008-01-01

    Embryonic stem cells need to maintain genomic integrity so that they can retain the ability to differentiate into multiple cell types without propagating DNA errors. Previous studies have suggested that mechanisms of genome surveillance, including DNA repair, are superior in mouse embryonic stem...... cells compared with various differentiated murine cells. Using single-cell gel electrophoresis (comet assay) we found that human embryonic stem cells (BG01, I6) have more efficient repair of different types of DNA damage (generated from H2O2, UV-C, ionizing radiation, or psoralen) than human primary...... fibroblasts (WI-38, hs27) and, with the exception of UV-C damage, HeLa cells. Microarray gene expression analysis showed that mRNA levels of several DNA repair genes are elevated in human embryonic stem cells compared with their differentiated forms (embryoid bodies). These data suggest that genomic...

  15. Restricted intra-embryonic origin of bona fide hematopoietic stem cells in the chicken

    NARCIS (Netherlands)

    Yvernogeau, Laurent; Robin, Catherine

    2017-01-01

    Hematopoietic stem cells (HSCs), which are responsible for blood cell production, are generated during embryonic development. Human and chicken embryos share features that position the chicken as a reliable and accessible alternative model to study developmental hematopoiesis. However, the existence

  16. Assessment of Stem Cell Markers During Long-Term Culture of Mouse Embryonic Stem Cells

    OpenAIRE

    Berrill, A.; Tan, H L; Wuang, S.C.; Fong, W.J.; Choo, Andre B. H.; Oh, Steve K. W.

    2004-01-01

    Embryonic stem (ES) cells have been in the fore front of scientific literature lately as having the potential for regeneration of many tissue types. Two important issues that need to be addressed are the culture conditions for maintaining ES cells and the accuracy of ES cell markers in monitoring the undifferentiated state. Leukaemia inhibitory factor (LIF) is routinely used to sustain mouse ES cells (mES) in a pluripotent fashion. In this paper, we assessed three markers during long-term mai...

  17. Selenite benefits embryonic stem cells therapy in Parkinson's disease.

    Science.gov (United States)

    Tian, L-P; Zhang, S; Xu, L; Li, W; Wang, Y; Chen, S-D; Ding, J-Q

    2012-09-01

    Embryonic stem cells (ESC) transplantation is a potential therapeutic approach for Parkinson's disease (PD). However, one of the main challenges to this therapy is the post-transplantation survival of dopaminergic (DA) neurons. In this study, mouse ESC were differentiated into DA neurons by a modified serum free protocol. These ESC-derived neurons were then transplanted into striatum of 6-OHDA lesioned rat. The viability of grafted DA neurons was decreased, accompanied by activated microglia and high levels of proinflammatory factors, such as TNF-α and iNOS, in the graft niche. This suggested that the local neuroinflammation might be involved in the reduced cells viability. Selenite, the source of essential micronutrient selenium, could inhibit NF-κB p65 nuclear translocation and subsequently reduce iNOS, COX-2 and TNF-α expression in LPS-treated BV2 cells in a dose dependant manner. Before the transplantation of ESC-derived DA neurons, 6-OHDA lesioned rats were intraperitoneally injected with selenite. The expression levels of TNF-α and iNOS were decreased by 30% and 50%, respectively, in selenite treated group. The survival of implanted DA neurons and the rotational behavior of transplanted rats were also remarkably improved by selenite treatment. To sum up, selenite might benefit ESCs transplantation therapy in PD through anti-inflammation effects.

  18. Gene delivery to rat and human Schwann cells and nerve segments: a comparison of AAV 1-9 and lentiviral vectors.

    Science.gov (United States)

    Hoyng, S A; De Winter, F; Gnavi, S; van Egmond, L; Attwell, C L; Tannemaat, M R; Verhaagen, J; Malessy, M J A

    2015-10-01

    Schwann cells (SCs) in an injured peripheral nerve form pathways for regenerating axons. Although these cells initially support regeneration, SCs lose their pro-regenerative properties following a prolonged period of denervation. Gene transfer to SC can enhance their therapeutic potential. In this article, we compared adeno-associated viral (AAV) vectors based on serotypes 1-9 for their capability to transduce cultured primary rat and human SCs and nerve segments. AAV1 is the best serotype to transduce rat SCs, whereas AAV2 and AAV6 performed equally well in human SCs. Transduction of monolayers of cultured rat and human SCs did not accurately predict the transduction efficiency in nerve segments. Rat nerve segments could be genetically modified equally well by a set of four AAV vectors (AAV1, AAV5, AAV7, AAV9), whereas AAV2 was superior in human nerve segments. The current experiments were undertaken as a first step towards future clinical implementation of ex vivo AAV-based gene therapy in surgical nerve repair. The transduction of rat and human SCs and nerve segments by entirely different AAV serotypes, as documented here, highlights one of the challenges of translating gene therapy from experimental animals to human patients.

  19. Characterization and comparison of embryonic stem cell-derived KDR+ cells with endothelial cells.

    Science.gov (United States)

    Sun, Xuan; Cheng, Lamei; Duan, Huaxin; Lin, Ge; Lu, Guangxiu

    2012-09-01

    Growing interest in utilizing endothelial cells (ECs) for therapeutic purposes has led to the exploration of human embryonic stem cells (hESCs) as a potential source for endothelial progenitors. In this study, ECs were induced from hESC lines and their biological characteristics were analyzed and compared with both cord blood endothelial progenitor cells (CBEPCs) and human umbilical vein endothelial cells (HUVECs) in vitro. The results showed that isolated embryonic KDR+ cells (EC-KDR+) display characteristics that were similar to CBEPCs and HUVECs. EC-KDR+, CBEPCs and HUVECs all expressed CD31 and CD144, incorporated DiI-Ac-LDL, bound UEA1 lectin, and were able to form tube-like structures on Matrigel. Compared with CBEPCs and HUVECs, the expression level of endothelial progenitor cell markers such as CD133 and KDR in EC-KDR+ was significantly higher, while the mature endothelial marker vWF was lowly expressed in EC-KDR+. In summary, the study showed that EC-KDR+ are primitive endothelial-like progenitors and might be a potential source for therapeutic vascular regeneration and tissue engineering. Copyright © 2012 Elsevier Inc. All rights reserved.

  20. File list: NoD.Emb.50.AllAg.Embryonic_germ_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Emb.50.AllAg.Embryonic_germ_cells mm9 No description Embryo Embryonic germ cell...36513,SRX335426,SRX336255 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Emb.50.AllAg.Embryonic_germ_cells.bed ...

  1. File list: InP.Emb.50.AllAg.Embryonic_germ_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Emb.50.AllAg.Embryonic_germ_cells mm9 Input control Embryo Embryonic germ cells... SRX272024 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Emb.50.AllAg.Embryonic_germ_cells.bed ...

  2. File list: InP.Emb.10.AllAg.Embryonic_germ_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Emb.10.AllAg.Embryonic_germ_cells mm9 Input control Embryo Embryonic germ cells... SRX272024 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Emb.10.AllAg.Embryonic_germ_cells.bed ...

  3. File list: InP.Emb.05.AllAg.Embryonic_germ_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Emb.05.AllAg.Embryonic_germ_cells mm9 Input control Embryo Embryonic germ cells... SRX272024 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Emb.05.AllAg.Embryonic_germ_cells.bed ...

  4. File list: NoD.Emb.10.AllAg.Embryonic_germ_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Emb.10.AllAg.Embryonic_germ_cells mm9 No description Embryo Embryonic germ cell...35439,SRX336513,SRX336255 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Emb.10.AllAg.Embryonic_germ_cells.bed ...

  5. 78 FR 25091 - Submission for OMB Review; 30-Day Comment Request: Request for Human Embryonic Stem Cell Line To...

    Science.gov (United States)

    2013-04-29

    ... for Human Embryonic Stem Cell Line To Be Approved for Use in NIH-Funded Research SUMMARY: Under the... be requested in writing. Proposed Collection: Request for Human Embryonic Stem Cell Line to be... used by applicants to request that human embryonic stem cell lines be approved for use in NIH-funded...

  6. File list: InP.Emb.20.AllAg.Embryonic_germ_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Emb.20.AllAg.Embryonic_germ_cells mm9 Input control Embryo Embryonic germ cells... SRX272024 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Emb.20.AllAg.Embryonic_germ_cells.bed ...

  7. File list: NoD.Emb.20.AllAg.Embryonic_germ_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Emb.20.AllAg.Embryonic_germ_cells mm9 No description Embryo Embryonic germ cell...36513,SRX336524,SRX336255 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Emb.20.AllAg.Embryonic_germ_cells.bed ...

  8. File list: NoD.Emb.05.AllAg.Embryonic_germ_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Emb.05.AllAg.Embryonic_germ_cells mm9 No description Embryo Embryonic germ cell...36256,SRX336475,SRX336255 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Emb.05.AllAg.Embryonic_germ_cells.bed ...

  9. Neural Progenitor Cells Derived from Human Embryonic Stem Cells as an Origin of Dopaminergic Neurons

    Directory of Open Access Journals (Sweden)

    Parinya Noisa

    2015-01-01

    Full Text Available Human embryonic stem cells (hESCs are able to proliferate in vitro indefinitely without losing their ability to differentiate into multiple cell types upon exposure to appropriate signals. Particularly, the ability of hESCs to differentiate into neuronal subtypes is fundamental to develop cell-based therapies for several neurodegenerative disorders, such as Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease. In this study, we differentiated hESCs to dopaminergic neurons via an intermediate stage, neural progenitor cells (NPCs. hESCs were induced to neural progenitor cells by Dorsomorphin, a small molecule that inhibits BMP signalling. The resulting neural progenitor cells exhibited neural bipolarity with high expression of neural progenitor genes and possessed multipotential differentiation ability. CBF1 and bFGF responsiveness of these hES-NP cells suggested their similarity to embryonic neural progenitor cells. A substantial number of dopaminergic neurons were derived from hES-NP cells upon supplementation of FGF8 and SHH, key dopaminergic neuron inducers. Importantly, multiple markers of midbrain neurons were detected, including NURR1, PITX3, and EN1, suggesting that hESC-derived dopaminergic neurons attained the midbrain identity. Altogether, this work underscored the generation of neural progenitor cells that retain the properties of embryonic neural progenitor cells. These cells will serve as an unlimited source for the derivation of dopaminergic neurons, which might be applicable for treating patients with Parkinson’s disease.

  10. Transforming pluripotency: an exon-level study of malignancy-specific transcripts in human embryonal carcinoma and embryonic stem cells.

    Science.gov (United States)

    Alagaratnam, Sharmini; Harrison, Neil; Bakken, Anne Cathrine; Hoff, Andreas M; Jones, Mark; Sveen, Anita; Moore, Harry D; Andrews, Peter W; Lothe, Ragnhild A; Skotheim, Rolf I

    2013-04-01

    To circumvent difficulties of isolating pure populations of cancer stem cells (CSCs) for the purpose of identifying malignancy-specific gene expression, we have compared exon-resolution transcriptomic profiles of 5 embryonal carcinoma (EC) cell lines, a histological subtype of germ cell tumor (GCT), to their nonmalignant caricature, specifically 6 human embryonic stem (ES) cell lines. Both cell types are readily accessible, and were purified for undifferentiated cells only. We identified a set of 28 differentially expressed genes, many of which had cancer and stemness roles. Overexpression of the recently discovered pluripotency gene NR5A2 in malignant EC cells revealed an intriguing indication of how WNT-mediated dysregulation of pluripotency is involved with malignancy. Expression of these 28 genes was further explored within 2 publically available data sets of primary EC tumors and normal testis. At the exon-level, alternative splicing events were detected in ZNF195, DNMT3B, and PMF1, and alternative promoters were detected for ASH2L and ETV5. These events were validated by reverse transcriptase-polymerase chain reaction-based methods in EC and ES lines, where the alternative splicing event in the de novo DNA methyltransferase DNMT3B may have functional consequences. In conclusion, we have identified malignancy-specific gene expression differences within a rigorous pluripotent stem cell context. These findings are of particular interest for both GCT and ES cell biology, and, in general, to the concept of CSCs.

  11. Development of buffalo (Bubalus bubalis embryonic stem cell lines from somatic cell nuclear transferred blastocysts

    Directory of Open Access Journals (Sweden)

    Syed Mohmad Shah

    2015-11-01

    Full Text Available We developed buffalo embryonic stem cell lines from somatic cell nuclear transfer derived blastocysts, produced by hand-guided cloning technique. The inner cell mass of the blastocyst was cut mechanically using a Microblade and cultured onto feeder cells in buffalo embryonic stem (ES cell culture medium at 38 °C in a 5% CO2 incubator. The stem cell colonies were characterized for alkaline phosphatase activity, karyotype, pluripotency and self-renewal markers like OCT4, NANOG, SOX2, c-Myc, FOXD3, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 and CD90. The cell lines also possessed the capability to differentiate across all the three germ layers under spontaneous differentiation conditions.

  12. Gro/TLE enables embryonic stem cell differentiation by repressing pluripotent gene expression

    DEFF Research Database (Denmark)

    Laing, Adam F; Lowell, Sally; Brickman, Joshua M

    2015-01-01

    Gro/TLE proteins (TLE1-4) are a family of transcriptional corepressors acting downstream of multiple signalling pathways. Several TLEs are expressed in a dynamic manner throughout embryonic development and at high levels in embryonic stem cells (ESCs). Here we find that Gro/TLE is not required...

  13. Derivation of keratinocytes from chicken embryonic stem cells: Establishment and characterization of differentiated proliferative cell populations

    Directory of Open Access Journals (Sweden)

    Mathilde Couteaudier

    2015-03-01

    Full Text Available A common challenge in avian cell biology is the generation of differentiated cell-lines, especially in the keratinocyte lineage. Only a few avian cell-lines are available and very few of them show an interesting differentiation profile. During the last decade, mammalian embryonic stem cell-lines were shown to differentiate into almost all lineages, including keratinocytes. Although chicken embryonic stem cells had been obtained in the 1990s, few differentiation studies toward the ectodermal lineage were reported. Consequently, we explored the differentiation of chicken embryonic stem cells toward the keratinocyte lineage by using a combination of stromal induction, ascorbic acid, BMP4 and chicken serum. During the induction period, we observed a downregulation of pluripotency markers and an upregulation of epidermal markers. Three homogenous cell populations were derived, which were morphologically similar to chicken primary keratinocytes, displaying intracellular lipid droplets in almost every pavimentous cell. These cells could be serially passaged without alteration of their morphology and showed gene and protein expression profiles of epidermal markers similar to chicken primary keratinocytes. These cells represent an alternative to the isolation of chicken primary keratinocytes, being less cumbersome to handle and reducing the number of experimental animals used for the preparation of primary cells.

  14. Epigenetic stability, adaptability, and reversibility in human embryonic stem cells.

    Science.gov (United States)

    Tompkins, Joshua D; Hall, Christine; Chen, Vincent Chang-yi; Li, Arthur Xuejun; Wu, Xiwei; Hsu, David; Couture, Larry A; Riggs, Arthur D

    2012-07-31

    The stability of human embryonic stem cells (hESCs) is of critical importance for both experimental and clinical applications. We find that as an initial response to altered culture conditions, hESCs change their transcription profile for hundreds of genes and their DNA methylation profiles for several genes outside the core pluripotency network. After adaption to conditions of feeder-free defined and/or xeno-free culture systems, expression and DNA methylation profiles are quite stable for additional passaging. However, upon reversion to the original feeder-based culture conditions, numerous transcription changes are not reversible. Similarly, although the majority of DNA methylation changes are reversible, highlighting the plasticity of DNA methylation, a few are persistent. Collectively, this indicates these cells harbor a memory of culture history. For culture-induced DNA methylation changes, we also note an intriguing correlation: hypomethylation of regions 500-2440 bp upstream of promoters correlates with decreased expression, opposite to that commonly seen at promoter-proximal regions. Lastly, changes in regulation of G-coupled protein receptor pathways provide a partial explanation for many of the unique transcriptional changes observed during hESC adaptation and reverse adaptation.

  15. Femtosecond optical transfection as a tool for genetic manipulation of human embryonic stem cells

    Science.gov (United States)

    Torres-Mapa, M. L.; Gardner, J.; Bradburn, H.; King, J.; Dholakia, K.; Gunn-Moore, F.

    2013-03-01

    We demonstrate the use of femtosecond optical transfection for the genetic manipulation of human embryonic stem cells. Using a system with an SLM combined with a scanning mirror allows poration of both single-cell and colony-formed human embryonic stem cells in a rapid and targeted manner. In this work, we show successful transfection of plasmid DNA tagged with fluorescent reporters into human embryonic stem cells using three doses of focused femtosecond laser. A significant number of transfected cells retained their undifferentiated morphological feature of large nucleus with high nucleus to cytoplasmic ratio, 48h after photoporation. Furthermore, DNA constructs driven by different types of promoters were also successfully transfected into human embryonic stem cells using this technique.

  16. Primed Pluripotent Cell Lines Derived from Various Embryonic Origins and Somatic Cells in Pig

    OpenAIRE

    Jin-Kyu Park; Hye-Sun Kim; Kyung-Jun Uh; Kwang-Hwan Choi; Hyeong-Min Kim; Taeheon Lee; Byung-Chul Yang; Hyun-Jong Kim; Hak-Hyun Ka; Heebal Kim; Chang-Kyu Lee

    2013-01-01

    Since pluripotent embryonic stem cell (ESC) lines were first derived from the mouse, tremendous efforts have been made to establish ESC lines in several domestic species including the pig; however, authentic porcine ESCs have not yet been established. It has proven difficult to maintain an ESC-like state in pluripotent porcine cell lines due to the frequent occurrence of spontaneous differentiation into an epiblast stem cell (EpiSC)-like state during culture. We have been able to derive EpiSC...

  17. Transplantation of Schwann Cells Inside PVDF-TrFE Conduits to Bridge Transected Rat Spinal Cord Stumps to Promote Axon Regeneration Across the Gap.

    Science.gov (United States)

    Lee, Yee-Shuan; Wu, Siliang; Arinzeh, Treena Livingston; Bunge, Mary Bartlett

    2017-11-03

    Among various models for spinal cord injury in rats, the contusion model is the most often used because it is the most common type of human spinal cord injury. The complete transection model, although not as clinically relevant as the contusion model, is the most rigorous method to evaluate axon regeneration. In the contusion model, it is difficult to distinguish regenerated from sprouted or spared axons due to the presence of remaining tissue post injury. In the complete transection model, a bridging method is necessary to fill the gap and create continuity from the rostral to the caudal stumps in order to evaluate the effectiveness of the treatments. A reliable bridging surgery is essential to test outcome measures by reducing the variability due to the surgical method. The protocols described here are used to prepare Schwann cells (SCs) and conduits prior to transplantation, complete transection of the spinal cord at thoracic level 8 (T8), insert the conduit, and transplant SCs into the conduit. This approach also uses in situ gelling of an injectable basement membrane matrix with SC transplantation that allows improved axon growth across the rostral and caudal interfaces with the host tissue.

  18. Contested embryonic culture in Japan--public discussion, and human embryonic stem cell research in an aging welfare society.

    Science.gov (United States)

    Sleeboom-Faulkner, Margaret

    2010-01-01

    This article explores the reasons for the lack of a broad discussion on bioethical regulation of human embryonic stem cell research (hESR) in Japan and asks why scientists experience difficulties accessing resources for hESR despite the acclaimed indifference of dominant Japanese culture to embryo research. The article shows how various social actors express their views on the embryo and oocyte donation in terms of dominant Japanese culture, foiled against what is regarded as Western culture. Second, it shows how the lack of concern with hESR should be understood in the context of public health policies and communications and bioethics decision making in Japan. Finally, it interprets the meaning of the embryo in the context of Japan as an aging modern welfare society, explaining how policymakers have come to emphasize the urgency of infertility problems over issues around abortion and embryonic life.

  19. Mechanism of arsenite toxicity in embryonic stem cells.

    Science.gov (United States)

    Beeravolu, Naimisha; McKee, Christina; Chaudhry, G Rasul

    2017-10-01

    Environmental arsenite exposure has been linked to cancer as well as other diseases, presenting an important and serious public health problem. Toxicity of inorganic arsenite (iAs) has been investigated using animal models and cell culture, yet its developmental effects are poorly understood. This study investigated the molecular mechanism of iAs toxicity to ascertain insight into development and differentiation processes using mouse embryonic stem cells (ESCs). The results showed that iAs exposure affected morphology and integrity of ESC colonies as well as inhibited cell growth in a concentration-dependent manner, excluding concentrations <1 μM iAs which stimulated ESC growth. ESCs self-renewal and pluripotency was also affected as evident from the downregulation of transcription circuitry, Oct4, Nanog, Sox2 and Klf4 resulting in non-specific differentiation. ESCs exposed to iAs randomly differentiated into three germ layers, mesoderm, endoderm and ectoderm, as judged by transcriptional expression of Brachyury, Gata4 and FGF2, as well as translational expression of BRACHYURY, GATA4 and TUJ1 respectively. The differentiated cells represented osteogenic, chondrogenic, myogenic and neurogenic lineages as evident from upregulation of Col1, Sox9, Col2, Myog, Notch, Nes and Nef. Although iAs caused slight apoptosis with a concomitant increase in ROS levels, the exposed ESCs had significant Bcl2 expression, which could be involved in the protection against apoptosis. Further analysis revealed upregulation of Jun and P38 in ESCs with an increase in iAs concentration. These observations indicated that iAs stress caused random differentiation of ESCs via JNK/P38 pathways. These findings suggest that iAs exposure may cause teratogenicity during early fetal development. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  20. NANOG reporter cell lines generated by gene targeting in human embryonic stem cells

    DEFF Research Database (Denmark)

    Fischer, Yvonne; Ganic, Elvira; Ameri, Jacqueline

    2010-01-01

    Pluripotency and self-renewal of human embryonic stem cells (hESCs) is mediated by a complex interplay between extra- and intracellular signaling pathways, which regulate the expression of pluripotency-specific transcription factors. The homeodomain transcription factor NANOG plays a central role...

  1. Human embryonic stem cells differentiate into functional renal proximal tubular-like cells.

    Science.gov (United States)

    Narayanan, Karthikeyan; Schumacher, Karl M; Tasnim, Farah; Kandasamy, Karthikeyan; Schumacher, Annegret; Ni, Ming; Gao, Shujun; Gopalan, Began; Zink, Daniele; Ying, Jackie Y

    2013-04-01

    Renal cells are used in basic research, disease models, tissue engineering, drug screening, and in vitro toxicology. In order to provide a reliable source of human renal cells, we developed a protocol for the differentiation of human embryonic stem cells into renal epithelial cells. The differentiated stem cells expressed markers characteristic of renal proximal tubular cells and their precursors, whereas markers of other renal cell types were not expressed or expressed at low levels. Marker expression patterns of these differentiated stem cells and in vitro cultivated primary human renal proximal tubular cells were comparable. The differentiated stem cells showed morphological and functional characteristics of renal proximal tubular cells, and generated tubular structures in vitro and in vivo. In addition, the differentiated stem cells contributed in organ cultures for the formation of simple epithelia in the kidney cortex. Bioreactor experiments showed that these cells retained their functional characteristics under conditions as applied in bioartificial kidneys. Thus, our results show that human embryonic stem cells can differentiate into renal proximal tubular-like cells. Our approach would provide a source for human renal proximal tubular cells that are not affected by problems associated with immortalized cell lines or primary cells.

  2. Data for whole and mitochondrial proteome of human embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Faezeh Shekari

    2017-08-01

    Full Text Available The data presented here pertain to the research article entitled “Proteome Analysis of Human Embryonic Stem Cell Organelles” (Shekariet al., 2017 [1]. In the present article we endeavour to locate new proteins and pathways in human embryonic stem cells (hESCs by mass spectrometry and bioinformatics analysis. We have analyzed total and mitochondrial proteins extracted from three biological replicates of the hESC H9 cell line according to mass spectrometry proteomics and bioinformatics investigations.

  3. Functional effect of mouse embryonic stem cell implantation after spinal cord injury

    OpenAIRE

    Lee, Tae-Hoon

    2013-01-01

    We transplanted mouse embryonic stem cells (mESCs) to improve functional loss in a rat model of clip-compression spinal cord injury (SCI). The mouse embryonic stem cells were transplanted to injured cord 7 days after injury. We include minimizing the progression of secondary injury, manipulating the neuroinhibitory environment of the spinal cord, replacing lost tissue with transplanted cells and substantial improvement of motor. A number of potential approaches optimize functional recovery af...

  4. Totipotent Embryonic Stem Cells Arise in Ground-State Culture Conditions

    DEFF Research Database (Denmark)

    Morgani, Sophie M; Canham, Maurice A; Nichols, Jennifer

    2013-01-01

    Embryonic stem cells (ESCs) are derived from mammalian embryos during the transition from totipotency, when individual blastomeres can make all lineages, to pluripotency, when they are competent to make only embryonic lineages. ESCs maintained with inhibitors of MEK and GSK3 (2i) are thought...... not directly support Nanog-positive epiblast-like ESCs. Thus, 2i and LIF support a totipotent state comparable to early embryonic cells that coexpress embryonic and extraembryonic determinants....... to represent an embryonically restricted ground state. However, we observed heterogeneous expression of the extraembryonic endoderm marker Hex in 2i-cultured embryos, suggesting that 2i blocked development prior to epiblast commitment. Similarly, 2i ESC cultures were heterogeneous and contained a Hex...

  5. Metastable primordial germ cell-like state induced from mouse embryonic stem cells by Akt activation

    Energy Technology Data Exchange (ETDEWEB)

    Yamano, Noriko [Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Kimura, Tohru, E-mail: tkimura@patho.med.osaka-u.ac.jp [Department of Pathology, Medical School, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Watanabe-Kushima, Shoko [Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Shinohara, Takashi [Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto 606-8501 (Japan); Nakano, Toru, E-mail: tnakano@patho.med.osaka-u.ac.jp [Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Department of Pathology, Medical School, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan)

    2010-02-12

    Specification to primordial germ cells (PGCs) is mediated by mesoderm-induction signals during gastrulation. We found that Akt activation during in vitro mesodermal differentiation of embryonic stem cells (ESCs) generated self-renewing spheres with differentiation states between those of ESCs and PGCs. Essential regulators for PGC specification and their downstream germ cell-specific genes were expressed in the spheres, indicating that the sphere cells had commenced differentiation to the germ lineage. However, the spheres did not proceed to spermatogenesis after transplantation into testes. Sphere cell transfer to the original feeder-free ESC cultures resulted in chaotic differentiation. In contrast, when the spheres were cultured on mouse embryonic fibroblasts or in the presence of ERK-cascade and GSK3 inhibitors, reversion to the ESC-like state was observed. These results indicate that Akt signaling promotes a novel metastable and pluripotent state that is intermediate to those of ESCs and PGCs.

  6. CD44 is expressed in non-myelinating Schwann cells of the adult rat, and may play a role in neurodegeneration-induced glial plasticity at the neuromuscular junction.

    Science.gov (United States)

    Gorlewicz, Adam; Wlodarczyk, Jakub; Wilczek, Ewa; Gawlak, Maciej; Cabaj, Anna; Majczynski, Henryk; Nestorowicz, Klaudia; Herbik, Magdalena Aneta; Grieb, Pawel; Slawinska, Urszula; Kaczmarek, Leszek; Wilczynski, Grzegorz M

    2009-05-01

    CD44 is a multifunctional cell surface glycoprotein which regulates cell-cell and cell-matrix interactions in a variety of tissues. In particular, the protein was found to be expressed in glial cells of developing, but not adult, peripheral nerves, where it takes part in signaling mediated by ErbB class of receptors for neuregulins. Here, we demonstrate, using high resolution morphological methods, tissue fractionation and RT-PCR, that CD44 is strongly expressed in terminal Schwann cell (TSC) at the neuromuscular junction (NMJ) of the adult rat skeletal muscle. As CD44 is also expressed by Schwann cells of the non-myelinated Remak bundles of the proximal peripheral nerves, it appears to be a marker of non-myelinating Schwann cell subpopulation. The analysis of transgenic rats bearing a mutated superoxide-dismutase gene (SOD1(G93A)) causing familial amyotrophic lateral sclerosis (ALS) revealed that TSC activation and morphological plasticity at the NMJ, caused by ongoing denervation-reinnervation is associated with a strong increase in CD44 expression therein. Notably, CD44 immunoreactivity is present in fine axon-escheating processes of the glial cells that guide reinnervation. In addition, we found that both in normal and SOD1(G93A) muscle, CD44 expressed in TSC partially colocalizes with immunoreactivities of neuregulin receptors ErbB2 and ErbB3. The colocalization appears to reflect a physical interaction, as evidenced by co-immunoprecipitation and fluorescence resonance energy transfer (FRET) analysis between CD44 and ErbB3. Importantly, TSC activation upon ALS-like neurodegeneration results in significant increase in molecular proximity of CD44 and ErbB3, which may have an impact on glial plasticity at the NMJ.

  7. Diabetes-induced effects on cardiomyocytes in chick embryonic heart micromass and mouse embryonic D3 differentiated stem cells.

    Science.gov (United States)

    Mohammed, Omar J; Latif, Muhammad Liaque; Pratten, Margaret K

    2017-04-01

    Diabetes mellitus during pregnancy is a considerable medical challenge, since it is related to ‎augmented morbidity and mortality concerns for both the fetus ‎and the pregnant woman. Records show that the etiology of diabetic ‎embryopathy is complicated, as many teratological factors might be involved ‎in the mechanisms of diabetes mellitus-induced congenital malformation. ‎In this study, the potential cardiotoxic effect of hyperglycemia with hyperketonemia was investigated by using two in vitro models; primary chick embryonic cardiomyocytes and stem cell derived cardiomyocytes, where adverse effects were recorded in both systems. The cells were evaluated by changes in beating activity, cell activity, protein content, ROS production, DNA damage and differentiating stem cell migration. The diabetic formulae used produced an increase in DNA damage and a decline in cell migration in mouse embryonic stem cells. These results provide an additional insight into adverse effects during gestational diabetes mellitus and a recommendation for expectant mothers and maternity staff to monitor glycaemic levels months ahead of conception. This study also supports the recommendation of using antioxidants during pregnancy to prevent DNA damage by the production of ROS, which might result in heart defects as well as other developmental anomalies. Copyright © 2017. Published by Elsevier Inc.

  8. 78 FR 13688 - Proposed Collection; 60-Day Comment Request: Request for Human Embryonic Stem Cell Line To Be...

    Science.gov (United States)

    2013-02-28

    ... Human Embryonic Stem Cell Line To Be Approved for Use in NIH Funded Research SUMMARY: In compliance with... Information Collection: The form is used by applicants to request that human embryonic stem cell lines be... within 60 days of the date of this publication. Proposed Collection: Request for Human Embryonic Stem...

  9. Generative models: Human embryonic stem cells and multiple modeling relations.

    Science.gov (United States)

    Fagan, Melinda Bonnie

    2016-04-01

    Model organisms are at once scientific models and concrete living things. It is widely assumed by philosophers of science that (1) model organisms function much like other kinds of models, and (2) that insofar as their scientific role is distinctive, it is in virtue of representing a wide range of biological species and providing a basis for generalizations about those targets. This paper uses the case of human embryonic stem cells (hESC) to challenge both assumptions. I first argue that hESC can be considered model organisms, analogous to classic examples such as Escherichia coli and Drosophila melanogaster. I then discuss four contrasts between the epistemic role of hESC in practice, and the assumptions about model organisms noted above. These contrasts motivate an alternative view of model organisms as a network of systems related constructively and developmentally to one another. I conclude by relating this result to other accounts of model organisms in recent philosophy of science. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Identification and quantitation of morphological cell types in electrophoretically separated human embryonic kidney cell cultures

    Science.gov (United States)

    Williams, K. B.; Kunze, M. E.; Todd, P. W.

    1985-01-01

    Four major cell types were identified by phase microscopy in early passage human embryonic kidney cell cultures. They are small and large epithelioid, domed, and fenestrated cells. Fibroblasts are also present in some explants. The percent of each cell type changes with passage number as any given culture grows. As a general rule, the fraction of small epithelioid cells increases, while the fraction of fenestrated cells, always small, decreases further. When fibroblasts are present, they always increase in percentage of the total cell population. Electrophoretic separation of early passage cells showed that the domed cells have the highest electrophoretic mobility, fibroblasts have an intermediate high mobility, small epithelioid cells have a low mobility, broadly distributed, and fenestrated cells have the lowest mobility. All cell types were broadly distributed among electrophoretic subfractions, which were never pure but only enriched with respect to a given cell type.

  11. Human dental follicle cells express embryonic, mesenchymal and neural stem cells markers.

    Science.gov (United States)

    Lima, Rodrigo Lopes; Holanda-Afonso, Rosenilde Carvalho; Moura-Neto, Vivaldo; Bolognese, Ana Maria; DosSantos, Marcos Fabio; Souza, Margareth Maria

    2017-01-01

    This study was conducted to identify and characterize dental follicle stem cells (DFSCs) by analyzing expression of embryonic, mesenchymal and neural stem cells surface markers. Design Dental follicle cells (DFCs) were evaluated by immunocytochemistry using embryonic stem cells markers (OCT4 and SOX2), mesenchmal stem cells (MSCs) markers (Notch1, active Notch1, STRO, CD44, HLA-ABC, CD90), neural stem cells markers (Nestin and β-III-tubulin), neural crest stem cells (NCSCs) markers (p75 and HNK1) and a glial cells marker (GFAP). RT-PCR was performed to identify the expression of OCT4 and NANOG in DFCs and dental follicle tissue. Immunocytochemistry and RT-PCR analysis revealed that a significant proportion of the DFCs evaluated expressed human embryonic stem cells marker OCT4 (75%) whereas NANOG was weakly expressed. A considerable amount of MSCs (90%) expressed Notch1, STRO, CD44 and HLA-ABC. However, they were weakly positive for CD90. Moreover, it was possible to demonstrate that dental follicle contains a significant proportion of neural stem/progenitors cells, expressing β-III-tubulin (90%) and nestin (70%). Interestingly, immunocytochemistry showed DFCs positive for p75 (50%), HNK1 (cells. This is the first study reporting the presence of NCSCs and glial-like cells in the dental follicle. The results of the present study suggest the occurrence of heterogeneous populations of stem cells, particularly neural stem/progenitor cells, in the dental follicle, Therefore, the human dental follicle might be a promising source of adult stem cells for regenerative purposes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Bioenergetic Changes during Differentiation of Human Embryonic Stem Cells along the Hepatic Lineage

    DEFF Research Database (Denmark)

    Hopkinson, Branden M; Madsen, Claus Desler; Kalisz, Mark

    2017-01-01

    of embryonic origin differentiating along the hepatic lineage. Our study reveals especially the transition between hepatic specification and hepatic maturation as dependent on mitochondrial respiration and demonstrates that even though differentiating cells are primarily dependent on glycolysis until induction...

  13. Prion protein expression regulates embryonic stem cell pluripotency and differentiation.

    Directory of Open Access Journals (Sweden)

    Alberto Miranda

    2011-04-01

    Full Text Available Cellular prion protein (PRNP is a glycoprotein involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs. Although the physiological function of PRNP is largely unknown, its key role in prion infection has been extensively documented. This study examines the functionality of PRNP during the course of embryoid body (EB differentiation in mouse Prnp-null (KO and WT embryonic stem cell (ESC lines. The first feature observed was a new population of EBs that only appeared in the KO line after 5 days of differentiation. These EBs were characterized by their expression of several primordial germ cell (PGC markers until Day 13. In a comparative mRNA expression analysis of genes playing an important developmental role during ESC differentiation to EBs, Prnp was found to participate in the transcription of a key pluripotency marker such as Nanog. A clear switching off of this gene on Day 5 was observed in the KO line as opposed to the WT line, in which maximum Prnp and Nanog mRNA levels appeared at this time. Using a specific antibody against PRNP to block PRNP pathways, reduced Nanog expression was confirmed in the WT line. In addition, antibody-mediated inhibition of ITGB5 (integrin αvβ5 in the KO line rescued the low expression of Nanog on Day 5, suggesting the regulation of Nanog transcription by Prnp via this Itgb5. mRNA expression analysis of the PRNP-related proteins PRND (Doppel and SPRN (Shadoo, whose PRNP function is known to be redundant, revealed their incapacity to compensate for the absence of PRNP during early ESC differentiation. Our findings provide strong evidence for a relationship between Prnp and several key pluripotency genes and attribute Prnp a crucial role in regulating self-renewal/differentiation status of ESC, confirming the participation of PRNP during early embryogenesis.

  14. A protocol for embryonic stem cell derivation by somatic cell nuclear transfer into human oocytes

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Dieter Egli & Gloryn Chia ### Abstract Here we describe detailed methods that allowed us to derive embryonic stem cell lines by nuclear transfer of fibroblasts from a newborn and from a type 1 diabetic adult. The protocol is based on the insight that 1) agents for cell fusion can act as potent mediators of oocyte activation by compromising maintaining plasma membrane integrity; minimizing the concentration at which they are used, and at least transiently remove calcium from ...

  15. Collagen Type I Improves the Differentiation of Human Embryonic Stem Cells towards Definitive Endoderm

    DEFF Research Database (Denmark)

    Rasmussen, Camilla Holzmann; Petersen, Dorthe Roenn; Møller, Jonas Bech

    2015-01-01

    Human embryonic stem cells have the ability to generate all cell types in the body and can potentially provide an unlimited source of cells for cell replacement therapy to treat degenerative diseases such as diabetes. Current differentiation protocols of human embryonic stem cells towards insulin...... embryonic stem cells to the definitive endoderm lineage. The percentage of definitive endoderm cells after differentiation on collagen I and fibronectin was >85% and 65%, respectively. The cells on collagen I substrates displayed different morphology and gene expression during differentiation as assessed...... and consistent differentiation of stem cells to definitive endoderm. The results shed light on the importance of extracellular matrix proteins for differentiation and also points to a cost effective and easy method to improve differentiation....

  16. Progressing a human embryonic stem-cell-based regenerative medicine therapy towards the clinic

    OpenAIRE

    Whiting, Paul; Kerby, Julie; Coffey, Peter; da Cruz, Lyndon; McKernan, Ruth

    2015-01-01

    Since the first publication of the derivation of human embryonic stem cells in 1998, there has been hope and expectation that this technology will lead to a wave of regenerative medicine therapies with the potential to revolutionize our approach to managing certain diseases. Despite significant resources in this direction, the path to the clinic for an embryonic stem-cell-based regenerative medicine therapy has not proven straightforward, though in the past few years progress has been made. H...

  17. Design of super-elastic biodegradable scaffolds with longitudinally oriented microchannels and optimization of the channel size for Schwann cell migration

    Directory of Open Access Journals (Sweden)

    Koichiro Uto, Takanari Muroya, Michio Okamoto, Hiroyuki Tanaka, Tsuyoshi Murase, Mitsuhiro Ebara and Takao Aoyagi

    2012-01-01

    Full Text Available We newly designed super-elastic biodegradable scaffolds with longitudinally oriented microchannels for repair and regeneration of peripheral nerve defects. Four-armed poly(ε-caprolactone-co-D,L-lactides (P(CL-co-DLLAs were synthesized by ring-opening copolymerization of CL and DLLA from terminal hydroxyl groups of pentaerythritol, and acryloyl chloride was then reacted with the ends of the chains. The end-functionalized P(CL-co-DLLA was crosslinked in a cylindrical mold in the presence of longitudinally oriented silica fibers as the templates, which were later dissolved by hydrofluoric acid. The elastic moduli of the crosslinked P(CL-co-DLLAs were controlled between 10−1 and 102 MPa at 37 °C, depending on the composition. The scaffolds could be elongated to 700% of their original size without fracture or damage ('super-elasticity'. Scanning electron microscopy images revealed that well-defined and highly aligned multiple channels consistent with the mold design were produced in the scaffolds. Owing to their elastic nature, the microchannels in the scaffolds did not collapse when they were bent to 90°. To evaluate the effect of the channel diameter on Schwann cell migration, microchannels were also fabricated in transparent poly(dimethylsiloxane, allowing observation of cell migration. The migration speed increased with channel size, but the Young's modulus of the scaffold decreased as the channel diameter increased. These findings may serve as the basis for designing tissue-engineering scaffolds for nerve regeneration and investigating the effects of the geometrical and dimensional properties on axonal outgrowth.

  18. [Remyelination properties of human embryonic nerve cells in conditions of long-term culture].

    Science.gov (United States)

    Tsymbaliuk, V I; Vasyl'ieva, I H; Oleksenko, N P; Chopyk, N H; Tsiubko, O I; Halanta, O S

    2009-01-01

    We have examined the mitogenic and differentiation potential and remyelination properties of human embryonic nerve cells in culture. After 1 month of cultivation without differentiation agents CNP-positive cells (the mitotically-active precursors of oligodendrocytes) were expanded at 3,6 times. At the same time the amount of GalC-positive cells (mature oligodendrocytes) remained low. So, the remyelination properties of embryonic nerve cells can be explained by high concentration of oligodendrocytes precursors. Cell population after cultivation maintained the increased remyelination potential by increasing the number of CNP-positive cells that was confirmed by using experimental demyelination.

  19. The similarity between human embryonic stem cell-derived epithelial cells and ameloblast-lineage cells.

    Science.gov (United States)

    Zheng, Li-Wei; Linthicum, Logan; DenBesten, Pamela K; Zhang, Yan

    2013-03-01

    This study aimed to compare epithelial cells derived from human embryonic stem cells (hESCs) to human ameloblast-lineage cells (ALCs), as a way to determine their potential use as a cell source for ameloblast regeneration. Induced by various concentrations of bone morphogenetic protein 4 (BMP4), retinoic acid (RA) and lithium chloride (LiCl) for 7 days, hESCs adopted cobble-stone epithelial phenotype (hESC-derived epithelial cells (ES-ECs)) and expressed cytokeratin 14. Compared with ALCs and oral epithelial cells (OE), ES-ECs expressed amelogenesis-associated genes similar to ALCs. ES-ECs were compared with human fetal skin epithelium, human fetal oral buccal mucosal epithelial cells and human ALCs for their expression pattern of cytokeratins as well. ALCs had relatively high expression levels of cytokeratin 76, which was also found to be upregulated in ES-ECs. Based on the present study, with the similarity of gene expression with ALCs, ES-ECs are a promising potential cell source for regeneration, which are not available in erupted human teeth for regeneration of enamel.

  20. The similarity between human embryonic stem cell-derived epithelial cells and ameloblast-lineage cells

    Science.gov (United States)

    Zheng, Li-Wei; Linthicum, Logan; DenBesten, Pamela K; Zhang, Yan

    2013-01-01

    This study aimed to compare epithelial cells derived from human embryonic stem cells (hESCs) to human ameloblast-lineage cells (ALCs), as a way to determine their potential use as a cell source for ameloblast regeneration. Induced by various concentrations of bone morphogenetic protein 4 (BMP4), retinoic acid (RA) and lithium chloride (LiCl) for 7 days, hESCs adopted cobble-stone epithelial phenotype (hESC-derived epithelial cells (ES-ECs)) and expressed cytokeratin 14. Compared with ALCs and oral epithelial cells (OE), ES-ECs expressed amelogenesis-associated genes similar to ALCs. ES-ECs were compared with human fetal skin epithelium, human fetal oral buccal mucosal epithelial cells and human ALCs for their expression pattern of cytokeratins as well. ALCs had relatively high expression levels of cytokeratin 76, which was also found to be upregulated in ES-ECs. Based on the present study, with the similarity of gene expression with ALCs, ES-ECs are a promising potential cell source for regeneration, which are not available in erupted human teeth for regeneration of enamel. PMID:23538640

  1. Novel Method To Differentiate Human Embryonic Stem Cells Into Dopaminergic Nerve Cells | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    The National Institute on Drug Abuse's Development and Plasticity Section is seeking statements of capability or interest from parties interested in licensing opportunities to further develop, evaluate, or commercialize novel methods to differentiate human embryonic stem cells into dopaminergic nerve cells. The invention described here is a novel method of differentiating human embryonic stem cells (hESCs) into dopaminergic nerve cells, which is preferable to the currently available dopaminergic differentiation techniques.

  2. STELLA facilitates differentiation of germ cell and endodermal lineages of human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Patompon Wongtrakoongate

    Full Text Available Stella is a developmentally regulated gene highly expressed in mouse embryonic stem (ES cells and in primordial germ cells (PGCs. In human, the gene encoding the STELLA homologue lies on chromosome 12p, which is frequently amplified in long-term cultured human ES cells. However, the role played by STELLA in human ES cells has not been reported. In the present study, we show that during retinoic acid (RA-induced differentiation of human ES cells, expression of STELLA follows that of VASA, a marker of germline differentiation. By contrast, human embryonal carcinoma cells express STELLA at a higher level compared with both karyotypically normal and abnormal human ES cell lines. We found that over-expression of STELLA does not interfere with maintenance of the stem cell state of human ES cells, but following retinoic acid induction it leads to up-regulation of germline- and endodermal-associated genes, whereas neural markers PAX6 and NEUROD1 are down-regulated. Further, STELLA over-expression facilitates the differentiation of human ES cells into BE12-positive cells, in which the expression of germline- and endodermal-associated genes is enriched, and suppresses differentiation of the neural lineage. Taken together, this finding suggests a role for STELLA in facilitating germline and endodermal differentiation of human ES cells.

  3. Computational analysis of expression of human embryonic stem cell-associated signatures in tumors

    Directory of Open Access Journals (Sweden)

    Wang Xiaosheng

    2011-10-01

    Full Text Available Abstract Background The cancer stem cell model has been proposed based on the linkage between human embryonic stem cells and human cancer cells. However, the evidences supporting the cancer stem cell model remain to be collected. In this study, we extensively examined the expression of human embryonic stem cell-associated signatures including core genes, transcription factors, pathways and microRNAs in various cancers using the computational biology approach. Results We used the class comparison analysis and survival analysis algorithms to identify differentially expressed genes and their associated transcription factors, pathways and microRNAs among normal vs. tumor or good prognosis vs. poor prognosis phenotypes classes based on numerous human cancer gene expression data. We found that most of the human embryonic stem cell- associated signatures were frequently identified in the analysis, suggesting a strong linkage between human embryonic stem cells and cancer cells. Conclusions The present study revealed the close linkage between the human embryonic stem cell associated gene expression profiles and cancer-associated gene expression profiles, and therefore offered an indirect support for the cancer stem cell theory. However, many interest issues remain to be addressed further.

  4. Nuclear accumulation and activation of p53 in embryonic stem cells after DNA damage

    Directory of Open Access Journals (Sweden)

    Rolletschek Alexandra

    2009-06-01

    Full Text Available Abstract Background P53 is a key tumor suppressor protein. In response to DNA damage, p53 accumulates to high levels in differentiated cells and activates target genes that initiate cell cycle arrest and apoptosis. Since stem cells provide the proliferative cell pool within organisms, an efficient DNA damage response is crucial. Results In proliferating embryonic stem cells, p53 is localized predominantly in the cytoplasm. DNA damage-induced nuclear accumulation of p53 in embryonic stem cells activates transcription of the target genes mdm2, p21, puma and noxa. We observed bi-phasic kinetics for nuclear accumulation of p53 after ionizing radiation. During the first wave of nuclear accumulation, p53 levels were increased and the p53 target genes mdm2, p21 and puma were transcribed. Transcription of noxa correlated with the second wave of nuclear accumulation. Transcriptional activation of p53 target genes resulted in an increased amount of proteins with the exception of p21. While p21 transcripts were efficiently translated in 3T3 cells, we failed to see an increase in p21 protein levels after IR in embryonal stem cells. Conclusion In embryonic stem cells where (anti-proliferative p53 activity is not necessary, or even unfavorable, p53 is retained in the cytoplasm and prevented from activating its target genes. However, if its activity is beneficial or required, p53 is allowed to accumulate in the nucleus and activates its target genes, even in embryonic stem cells.

  5. Nuclear accumulation and activation of p53 in embryonic stem cells after DNA damage.

    Science.gov (United States)

    Solozobova, Valeriya; Rolletschek, Alexandra; Blattner, Christine

    2009-06-17

    P53 is a key tumor suppressor protein. In response to DNA damage, p53 accumulates to high levels in differentiated cells and activates target genes that initiate cell cycle arrest and apoptosis. Since stem cells provide the proliferative cell pool within organisms, an efficient DNA damage response is crucial. In proliferating embryonic stem cells, p53 is localized predominantly in the cytoplasm. DNA damage-induced nuclear accumulation of p53 in embryonic stem cells activates transcription of the target genes mdm2, p21, puma and noxa. We observed bi-phasic kinetics for nuclear accumulation of p53 after ionizing radiation. During the first wave of nuclear accumulation, p53 levels were increased and the p53 target genes mdm2, p21 and puma were transcribed. Transcription of noxa correlated with the second wave of nuclear accumulation. Transcriptional activation of p53 target genes resulted in an increased amount of proteins with the exception of p21. While p21 transcripts were efficiently translated in 3T3 cells, we failed to see an increase in p21 protein levels after IR in embryonal stem cells. In embryonic stem cells where (anti-proliferative) p53 activity is not necessary, or even unfavorable, p53 is retained in the cytoplasm and prevented from activating its target genes. However, if its activity is beneficial or required, p53 is allowed to accumulate in the nucleus and activates its target genes, even in embryonic stem cells.

  6. Melphalan, Carboplatin, Mannitol, and Sodium Thiosulfate in Treating Patients With Recurrent or Progressive CNS Embryonal or Germ Cell Tumors

    Science.gov (United States)

    2017-10-31

    Adult Central Nervous System Germ Cell Tumor; Adult Embryonal Tumor With Multilayered Rosettes, C19MC-Altered; Adult Medulloblastoma; Adult Pineoblastoma; Adult Supratentorial Embryonal Tumor, Not Otherwise Specified; Atypical Teratoid/Rhabdoid Tumor; Childhood Atypical Teratoid/Rhabdoid Tumor; Childhood Central Nervous System Germ Cell Tumor; Childhood Embryonal Tumor With Multilayered Rosettes, C19MC-Altered; Medulloepithelioma; Ototoxicity; Recurrent Adult Brain Neoplasm; Recurrent Childhood Central Nervous System Embryonal Neoplasm; Recurrent Childhood Malignant Germ Cell Tumor; Recurrent Childhood Medulloblastoma; Recurrent Childhood Pineoblastoma; Recurrent Childhood Supratentorial Embryonal Tumor, Not Otherwise Specified

  7. Effects of Pulsed Electromagnetic Field on Differentiation of HUES-17 Human Embryonic Stem Cell Line

    Directory of Open Access Journals (Sweden)

    Yi-Lin Wu

    2014-08-01

    Full Text Available Electromagnetic fields are considered to potentially affect embryonic development, but the mechanism is still unknown. In this study, human embryonic stem cell (hESC line HUES-17 was applied to explore the mechanism of exposure on embryonic development to pulsed electromagnetic field (PEMF for 400 pulses at different electric field intensities and the differentiation of HUES-17 cells was observed after PEMF exposure. The expression of alkaline phosphatase (AP, stage-specific embryonic antigen-3 (SSEA-3, SSEA-4 and the mRNA level and protein level of Oct4, Sox2 and Nanog in HUES-17 cells remained unchanged after PEMF exposure at the electric field intensities of 50, 100, 200 or 400 kV/m. Four hundred pulses PEMF exposure at the electric field intensities of 50, 100, 200 or 400 kV/m did not affect the differentiation of HUES-17 cells. The reason why electromagnetic fields affect embryonic development may be due to other mechanisms rather than affecting the differentiation of embryonic stem cells.

  8. Immunohistochemical, ultrastructural and functional analysis of axonal regeneration through peripheral nerve grafts containing Schwann cells expressing BDNF, CNTF or NT3.

    Directory of Open Access Journals (Sweden)

    Maria João Godinho

    Full Text Available We used morphological, immunohistochemical and functional assessments to determine the impact of genetically-modified peripheral nerve (PN grafts on axonal regeneration after injury. Grafts were assembled from acellular nerve sheaths repopulated ex vivo with Schwann cells (SCs modified to express brain-derived neurotrophic factor (BDNF, a secretable form of ciliary neurotrophic factor (CNTF, or neurotrophin-3 (NT3. Grafts were used to repair unilateral 1 cm defects in rat peroneal nerves and 10 weeks later outcomes were compared to normal nerves and various controls: autografts, acellular grafts and grafts with unmodified SCs. The number of regenerated βIII-Tubulin positive axons was similar in all grafts with the exception of CNTF, which contained the fewest immunostained axons. There were significantly lower fiber counts in acellular, untransduced SC and NT3 groups using a PanNF antibody, suggesting a paucity of large caliber axons. In addition, NT3 grafts contained the greatest number of sensory fibres, identified with either IB4 or CGRP markers. Examination of semi- and ultra-thin sections revealed heterogeneous graft morphologies, particularly in BDNF and NT3 grafts in which the fascicular organization was pronounced. Unmyelinated axons were loosely organized in numerous Remak bundles in NT3 grafts, while the BDNF graft group displayed the lowest ratio of umyelinated to myelinated axons. Gait analysis revealed that stance width was increased in rats with CNTF and NT3 grafts, and step length involving the injured left hindlimb was significantly greater in NT3 grafted rats, suggesting enhanced sensory sensitivity in these animals. In summary, the selective expression of BDNF, CNTF or NT3 by genetically modified SCs had differential effects on PN graft morphology, the number and type of regenerating axons, myelination, and locomotor function.

  9. Sciatic nerve regeneration by transplantation of Schwann cells via erythropoietin controlled-releasing polylactic acid/multiwalled carbon nanotubes/gelatin nanofibrils neural guidance conduit.

    Science.gov (United States)

    Salehi, Majid; Naseri-Nosar, Mahdi; Ebrahimi-Barough, Somayeh; Nourani, Mohammdreza; Khojasteh, Arash; Hamidieh, Amir-Ali; Amani, Amir; Farzamfar, Saeed; Ai, Jafar

    2017-07-04

    The current study aimed to enhance the efficacy of peripheral nerve regeneration using an electrically conductive biodegradable porous neural guidance conduit for transplantation of allogeneic Schwann cells (SCs). The conduit was produced from polylactic acid (PLA), multiwalled carbon nanotubes (MWCNTs), and gelatin nanofibrils (GNFs) coated with the recombinant human erythropoietin-loaded chitosan nanoparticles (rhEpo-CNPs). The PLA/MWCNTs/GNFs/rhEpo-CNPs conduit had the porosity of 85.78 ± 0.70%, the contact angle of 77.65 ± 1.91° and the ultimate tensile strength and compressive modulus of 5.51 ± 0.13 MPa and 2.66 ± 0.34 MPa, respectively. The conduit showed the electrical conductivity of 0.32 S cm(-1) and lost about 11% of its weight after 60 days in normal saline. The produced conduit was able to release the rhEpo for at least 2 weeks and exhibited favorable cytocompatibility towards SCs. For functional analysis, the conduit was seeded with 1.5 × 10(4) SCs and implanted into a 10 mm sciatic nerve defect of Wistar rat. After 14 weeks, the results of sciatic functional index, hot plate latency, compound muscle action potential amplitude, weight-loss percentage of wet gastrocnemius muscle and Histopathological examination using hematoxylin-eosin and Luxol fast blue staining demonstrated that the produced conduit had comparable nerve regeneration to the autograft, as the gold standard to bridge the nerve gaps. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2017. © 2017 Wiley Periodicals, Inc.

  10. β-Cell Replacement in Mice Using Human Type 1 Diabetes Nuclear Transfer Embryonic Stem Cells.

    Science.gov (United States)

    Sui, Lina; Danzl, Nichole; Campbell, Sean R; Viola, Ryan; Williams, Damian; Xing, Yuan; Wang, Yong; Phillips, Neil; Poffenberger, Greg; Johannesson, Bjarki; Oberholzer, Jose; Powers, Alvin C; Leibel, Rudolph L; Chen, Xiaojuan; Sykes, Megan; Egli, Dieter

    2018-01-01

    β-Cells derived from stem cells hold great promise for cell replacement therapy for diabetes. Here we examine the ability of nuclear transfer embryonic stem cells (NT-ESs) derived from a patient with type 1 diabetes to differentiate into β-cells and provide a source of autologous islets for cell replacement. NT-ESs differentiate in vitro with an average efficiency of 55% into C-peptide-positive cells, expressing markers of mature β-cells, including MAFA and NKX6.1. Upon transplantation in immunodeficient mice, grafted cells form vascularized islet-like structures containing MAFA/C-peptide-positive cells. These β-cells adapt insulin secretion to ambient metabolite status and show normal insulin processing. Importantly, NT-ES-β-cells maintain normal blood glucose levels after ablation of the mouse endogenous β-cells. Cystic structures, but no teratomas, were observed in NT-ES-β-cell grafts. Isogenic induced pluripotent stem cell lines showed greater variability in β-cell differentiation. Even though different methods of somatic cell reprogramming result in stem cell lines that are molecularly indistinguishable, full differentiation competence is more common in ES cell lines than in induced pluripotent stem cell lines. These results demonstrate the suitability of NT-ES-β-cells for cell replacement for type 1 diabetes and provide proof of principle for therapeutic cloning combined with cell therapy. © 2017 by the American Diabetes Association.

  11. Embryonic Stem Cell-Like Subpopulations in Venous Malformation

    Directory of Open Access Journals (Sweden)

    Elysia M. S. Tan

    2017-10-01

    Full Text Available BackgroundVenous malformation (VM consists of a network of ectatic anomalous thin-walled venous channels. A role for an activating TIE2 mutation in the development of the dilated luminal vessels in VM, and its proposed involvement of embryonic stem cells (ESCs, led us to investigate the expression of ESC markers in subcutaneous VM (SCVM and intramuscular VM (IMVM.MethodsFormalin-fixed paraffin-embedded sections of SCVM from seven patients and IMVM samples from seven patients were analyzed for the expression of Nanog, pSTAT3, OCT4, SOX2, SALL4, and CD44, using 3,3′-diaminobenzidine (DAB immunohistochemical (IHC staining. All these samples did not express lymphatic marker D2-40. NanoString mRNA analysis and RT-PCR were performed on snap-frozen samples of SCVM (n = 3 and IMVM (n = 3 from the respective original cohorts of patients included in DAB IHC staining. To confirm co-expression of two proteins, immunofluorescent (IF IHC staining on two representative samples of IMVM and SCVM samples from the original cohorts of patients included for DAB IHC staining was performed.ResultsDAB IHC staining demonstrated expression of all of the above ESC markers in both SCVM and IMVM samples. IF IHC staining showed that these markers were localized to the endothelium within these lesions and that Nanog, pSTAT3, SOX2, and CD44 were also expressed by cells outside of the endothelium. NanoString mRNA analysis confirmed transcription activation of pSTAT3, OCT4, and CD44. RT-qPCR confirmed transcription activation of Nanog, SOX2, and SALL4.ConclusionOur findings support the presence of two ESC-like subpopulations, one within and one outside of the endothelium, of both SCVM and IMVM. Given that the endothelial ESC-like subpopulation expresses the more primitive marker, OCT4, it is exciting to speculate that they give rise to the non-endothelial subpopulation.

  12. In vitro germ cell differentiation from cynomolgus monkey embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Kaori Yamauchi

    Full Text Available BACKGROUND: Mouse embryonic stem (ES cells can differentiate into female and male germ cells in vitro. Primate ES cells can also differentiate into immature germ cells in vitro. However, little is known about the differentiation markers and culture conditions for in vitro germ cell differentiation from ES cells in primates. Monkey ES cells are thus considered to be a useful model to study primate gametogenesis in vitro. Therefore, in order to obtain further information on germ cell differentiation from primate ES cells, this study examined the ability of cynomolgus monkey ES cells to differentiate into germ cells in vitro. METHODS AND FINDINGS: To explore the differentiation markers for detecting germ cells differentiated from ES cells, the expression of various germ cell marker genes was examined in tissues and ES cells of the cynomolgus monkey (Macaca fascicularis. VASA is a valuable gene for the detection of germ cells differentiated from ES cells. An increase of VASA expression was observed when differentiation was induced in ES cells via embryoid body (EB formation. In addition, the expression of other germ cell markers, such as NANOS and PIWIL1 genes, was also up-regulated as the EB differentiation progressed. Immunocytochemistry identified the cells expressing stage-specific embryonic antigen (SSEA 1, OCT-4, and VASA proteins in the EBs. These cells were detected in the peripheral region of the EBs as specific cell populations, such as SSEA1-positive, OCT-4-positive cells, OCT-4-positive, VASA-positive cells, and OCT-4-negative, VASA-positive cells. Thereafter, the effect of mouse gonadal cell-conditioned medium and growth factors on germ cell differentiation from monkey ES cells was examined, and this revealed that the addition of BMP4 to differentiating ES cells increased the expression of SCP1, a meiotic marker gene. CONCLUSION: VASA is a valuable gene for the detection of germ cells differentiated from ES cells in monkeys, and the

  13. Association of intratubular seminoma and intratubular embryonal carcinoma with invasive testicular germ cell tumors.

    Science.gov (United States)

    Lau, Sean K; Weiss, Lawrence M; Chu, Peiguo G

    2007-07-01

    The classification of intratubular germ cell neoplasia of the testis includes an unclassified type (IGCNU), in addition to various other intratubular lesions that show specific forms of differentiation, such as intratubular seminoma and intratubular embryonal carcinoma. Although IGCNU is recognized as a precursor lesion for testicular germ cell tumors, the relationship between differentiated types of intratubular germ cell neoplasia and invasive germ cell tumors of the testis is not well established. The aim of the present study was to examine the association between invasive testicular germ cell tumors and intratubular neoplastic lesions, with particular emphasis on differentiated types of intratubular germ cell neoplasia. The seminiferous tubules adjacent to 42 testicular germ cell tumors were evaluated for the presence of various forms of intratubular germ cell neoplasia. IGCNU was observed in 37 (88%) of 42 cases, whereas intratubular seminoma and intratubular embryonal carcinoma were seen in 19% and 7% of the cases, respectively. Intratubular seminoma was associated primarily with seminomas or mixed germ cell tumors with a seminomatous component, but was also present in a case of a nonseminomatous germ cell tumor. Intratubular embryonal carcinoma was associated exclusively with nonseminomatous germ cell tumors. All cases of intratubular embryonal carcinoma were identified morphologically and exhibited histologic features corresponding to traditional definitions of this lesion. No examples of intratubular embryonal carcinoma as defined by CD30 expression alone in the absence of an intratubular proliferation were observed. The presence of intratubular seminoma in a nonseminomatous germ cell tumor suggests that it is a true preinvasive lesion rather than a manifestation of intratubular spread of an established invasive seminoma. The low incidence of intratubular embryonal carcinoma supports the theory that most nonseminomatous germ cell tumors evolve initially as

  14. Embryonic stem cells improve skeletal muscle recovery after extreme atrophy in mice.

    Science.gov (United States)

    Artioli, Guilherme Giannini; De Oliveira Silvestre, João Guilherme; Guilherme, João Paulo Limongi França; Baptista, Igor Luchini; Ramos, Gracielle Vieira; Da Silva, Willian José; Miyabara, Elen Haruka; Moriscot, Anselmo Sigari

    2015-03-01

    We injected embryonic stem cells into mouse tibialis anterior muscles subjected to botulinum toxin injections as a model for reversible neurogenic atrophy. Muscles were exposed to botulinum toxin for 4 weeks and allowed to recover for up to 6 weeks. At the onset of recovery, a single muscle injection of embryonic stem cells was administered. The myofiber cross-sectional area, single twitch force, peak tetanic force, time-to-peak force, and half-relaxation time were determined. Although the stem cell injection did not affect the myofiber cross-sectional area gain in recovering muscles, most functional parameters improved significantly compared with those of recovering muscles that did not receive the stem cell injection. Muscle function recovery was accelerated by embryonic stem cell delivery in this durable neurogenic atrophy model. We conclude that stem cells should be considered a potential therapeutic tool for recovery after extreme skeletal muscle atrophy. © 2014 Wiley Periodicals, Inc.

  15. Cell surface carbohydrate changes during embryonic and fetal skin development

    DEFF Research Database (Denmark)

    Dabelsteen, Erik; Holbrook, K; Clausen, H

    1986-01-01

    Monoclonal antibodies to four type 2 chain carbohydrate antigens were used for immunohistochemical studies of embryonic and fetal skin. The antibodies detected N-acetyllactosamine and 3 fucosyl substitutes of this, blood group antigen H, Lex, and Ley. Periderm consistently stained for N...

  16. Properties and uses of embryonic stem cells: prospects for application to human biology and gene therapy.

    Science.gov (United States)

    Rathjen, P D; Lake, J; Whyatt, L M; Bettess, M D; Rathjen, J

    1998-01-01

    Embryonic stem cells are pluripotent cells derived from the early mouse embryo that can be propagated stably in the undifferentiated state in vitro. They retain the ability to differentiate into all cell types found in an embryonic and adult mouse in vivo, and can be induced to differentiate into many cell types in vitro. Exploitation of ES cell technology for the creation of mice bearing predetermined genetic alterations has received widespread attention because of the sophistication that it brings to the study of gene function in mammals. Analysis of cell differentiation in vitro has also been of value, leading to the identification of novel bioactive factors and the elucidation of cell specification mechanisms. In this paper, we summarise the features of pluripotent cell lines and their applications, foreshadowing the impact that these systems may have on human biology. While the isolation of definitive human pluripotent cell lines has not yet been achieved, potential applications for these cells in the study of human biology, particularly cell specification, can be envisaged. Of particular interest is the possibility that human embryonic stem cells with properties similar to mouse embryonic stem cells might provide a generic system for gene therapy.

  17. Isolation and characterization of node/notochord-like cells from mouse embryonic stem cells

    DEFF Research Database (Denmark)

    Winzi, Maria K.; Hyttel, Poul; Dale, Jacqueline Kim

    2011-01-01

    The homeobox gene Noto is expressed in the node and its derivative the notochord. Here we use a targeted Noto-GFP reporter to isolate and characterize node/notochord-like cells derived from mouse embryonic stem cells. We find very few Noto-expressing cells after spontaneous differentiation. However......, the number of Noto-expressing cells was increased when using Activin A to induce a Foxa2- and Brachyury-expressing progenitor population, whose further differentiation into Noto-expressing cells was improved by simultaneous inhibition of BMP, Wnt, and retinoic acid signaling. Noto-GFP(+) cells expressed...... the node/notochord markers Noto, Foxa2, Shh, Noggin, Chordin, Foxj1, and Brachyury; showed a vacuolarization characteristic of notochord cells; and can integrate into midline structures when grafted into Hensen's node of gastrulating chicken embryos. The ability to generate node/notochord-like cells...

  18. Chemical and physical factors influencing the dynamics of differentiation in embryonic stem cells.

    Science.gov (United States)

    Jyoti, Saras; Tandon, Simran

    2015-01-01

    Differentiating embryonic stem cells into a specific lineage or cell type is one of the most investigated areas of stem cell research; however it is wrought with hurdles. The differentiation process during embryogenesis is greatly influenced by physiochemical factors. They direct key genes for lineage commitment, which in turn are responsible for patterning into highly organized tissues resulting in organ formation. In this review, we have focused on the influence of physiochemical factors on the differentiation process in embryonic stem cells, which mimics, embryogenesis in vivo.

  19. Embryonic Stem Cell Culture Conditions Support Distinct States Associated with Different Developmental Stages and Potency

    DEFF Research Database (Denmark)

    Martin Gonzalez, Javier; Morgani, Sophie M; Bone, Robert A

    2016-01-01

    Embryonic stem cells (ESCs) are cell lines derived from the mammalian pre-implantation embryo. Here we assess the impact of derivation and culture conditions on both functional potency and ESC transcriptional identity. Individual ESCs cultured in either two small-molecule inhibitors (2i) or with ......Embryonic stem cells (ESCs) are cell lines derived from the mammalian pre-implantation embryo. Here we assess the impact of derivation and culture conditions on both functional potency and ESC transcriptional identity. Individual ESCs cultured in either two small-molecule inhibitors (2i...

  20. TET2 deficiency inhibits mesoderm and hematopoietic differentiation in human embryonic stem cells

    DEFF Research Database (Denmark)

    Langlois, Thierry; da Costa Reis Monte Mor, Barbara; Lenglet, Gaëlle

    2014-01-01

    . Here, we show that TET2 expression is low in human embryonic stem (ES) cell lines and increases during hematopoietic differentiation. ShRNA-mediated TET2 knockdown had no effect on the pluripotency of various ES cells. However, it skewed their differentiation into neuroectoderm at the expense...... profile, including abnormal expression of neuronal genes. Intriguingly, when TET2 was knockdown in hematopoietic cells, it increased hematopoietic development. In conclusion, our work suggests that TET2 is involved in different stages of human embryonic development, including induction of the mesoderm...... and hematopoietic differentiation. Stem Cells 2014....

  1. The role of nanotechnology in induced pluripotent and embryonic stem cells research.

    Science.gov (United States)

    Chen, Lukui; Qiu, Rong; Li, Lushen

    2014-12-01

    This paper reviews the recent studies on development of nanotechnology in the field of induced pluripotent and embryonic stem cells. Stem cell therapy is a promising therapy that can improve the quality of life for patients with refractory diseases. However, this option is limited by the scarcity of tissues, ethical problem, and tumorigenicity. Nanotechnology is another promising therapy that can be used to mimic the extracellular matrix, label the implanted cells, and also can be applied in the tissue engineering. In this review, we briefly introduce implementation of nanotechnology in induced pluripotent and embryonic stem cells research. Finally, the potential application of nanotechnology in tissue engineering and regenerative medicine is also discussed.

  2. Embryonic stem cells and prospects for their use in regenerative medicine approaches to motor neurone disease.

    Science.gov (United States)

    Christou, Y A; Moore, H D; Shaw, P J; Monk, P N

    2007-10-01

    Human embryonic stem cells are pluripotent cells with the potential to differentiate into any cell type in the presence of appropriate stimulatory factors and environmental cues. Their broad developmental potential has led to valuable insights into the principles of developmental and cell biology and to the proposed use of human embryonic stem cells or their differentiated progeny in regenerative medicine. This review focuses on the prospects for the use of embryonic stem cells in cell-based therapy for motor neurone disease or amyotrophic lateral sclerosis, a progressive neurodegenerative disease that specifically affects upper and lower motor neurones and leads ultimately to death from respiratory failure. Stem cell-derived motor neurones could conceivably be used to replace the degenerated cells, to provide authentic substrates for drug development and screening and for furthering our understanding of disease mechanisms. However, to reliably and accurately culture motor neurones, the complex pathways by which differentiation occurs in vivo must be understood and reiterated in vitro by embryonic stem cells. Here we discuss the need for new therapeutic strategies in the treatment of motor neurone disease, the developmental processes that result in motor neurone formation in vivo, a number of experimental approaches to motor neurone production in vitro and recent progress in the application of stem cells to the treatment and understanding of motor neurone disease.

  3. Development of heart muscle-cell diversity: a help or a hindrance for phenotyping embryonic stem cell-derived cardiomyocytes

    NARCIS (Netherlands)

    Fijnvandraat, Arnoud C.; Lekanne Deprez, Ronald H.; Moorman, Antoon F. M.

    2003-01-01

    Despite the advances in cardiovascular treatment, cardiac disease remains a major cause of morbidity in all industrialized countries. The extraordinary potential of (embryonic) stem cells for therapeutic purposes has revolutionized ideas about cardiac repair of diseased cardiac muscle to exciting

  4. Laser fusion of mouse embryonic cells and intra-embryonic fusion of blastomeres without affecting the embryo integrity.

    Directory of Open Access Journals (Sweden)

    Alexander Krivokharchenko

    Full Text Available Manipulation with early mammalian embryos is the one of the most important approach to study preimplantation development. Artificial cell fusion is a research tool for various biotechnological experiments. However, the existing methods have various disadvantages, first of them impossibility to fuse selected cells within multicellular structures like mammalian preimplantation embryos. In our experiments we have successfully used high repetition rate picosecond near infrared laser beam for fusion of pairs of oocytes and oocytes with blastomeres. Fused cells looked morphologically normal and keep their ability for further divisions in vitro. We also fused two or three blastomeres inside four-cell mouse embryos. The presence of one, two or three nuclei in different blastomeres of the same early preimplantation mouse embryo was confirmed under UV-light after staining of DNA with the vital dye Hoechst-33342. The most of established embryos demonstrated high viability and developed in vitro to the blastocyst stage. We demonstrated for the first time the use of laser beam for the fusion of various embryonic cells of different size and of two or three blastomeres inside of four-cell mouse embryos without affecting the embryo's integrity and viability. These embryos with blastomeres of various ploidy maybe unique model for numerous purposes. Thus, we propose laser optical manipulation as a new tool for investigation of fundamental mechanisms of mammalian development.

  5. Comparison of various types of collagenous scaffolds applied for embryonic nerve cell culture.

    Science.gov (United States)

    Drobnik, Jacek; Pietrucha, Krystyna; Kudzin, Marcin; Mader, Krystyna; Szymański, Jacek; Szczepanowska, Alicja

    2017-03-01

    The purpose of the study was to confirm whether collagen-based scaffolds using different cross-linking methods are suitable elaborate environments for embryonic nerve cell culture. Three 3D sponge-shaped porous scaffolds were composed using collagen alone, collagen with chondroitin sulphate modified by 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride, and collagen cross-linked by 2,3-dialdehyde cellulose (DAC). Embryonic nerve cells from rats were applied to the scaffolds and stained with bisbenzimide to study cell entrapment within the scaffolds. The metabolic activity of the cells cultured in the scaffolds was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The majority of cells were differentiated into neurocytes or oligodendrocytes. Collagen and collagen-chondroitin sulphate scaffolds entrapped a low number of cells. The highest cell density was found in the collagen-DAC scaffold. Moreover, in collagen-DAC scaffolds, the metabolic activity was markedly higher than in the other samples. Although all used scaffolds are suitable for the culture of embryonic nerve cells, the collagen-DAC scaffold properties are the most favorable. This scaffold entraps the highest number of cells and constitutes a favorable environment for their culture. Hence, the Col-DAC scaffold is recommended as an effective carrier for embryonic nerve cells. Copyright © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  6. Regulation of APC/C (Cdh1) ubiquitin ligase in differentiation of human embryonic stem cells.

    Science.gov (United States)

    Bar-On, Ortal; Shapira, Ma'anit; Skorecki, Karl; Hershko, Avram; Hershko, Dan D

    2010-05-15

    We have recently shown that Skp2 levels are high in undifferentiated human embryonic stem cells, but decline rapidly following induction of differentiation, thereby leading to accumulation of p27. Changes in Skp2 levels were found to be caused mainly by its rate of degradation. Here we show that the activity of APC/C (Cdh1), the ubiquitin ligase that targets Skp2 for degradation, increases markedly during the differentiation process of human embryonic stem cells. APC/C (Cdh1) is present but inactive in undifferentiated embryonic stem cells and becomes active in the differentiated state. The rise in APC/C (Cdh1) activity with differentiation appears to be due, at least in part, to a dramatic decline in the levels of its inhibitor Emi1. In addition, protein kinase activity also appears to contribute to the suppression of APC/C (Cdh1) activity in undifferentiated stem cells, possibly by inhibitory phosphorylation of Cdh1.

  7. Male Differentiation of Germ Cells Induced by Embryonic Age-Specific Sertoli Cells in Mice1

    Science.gov (United States)

    Ohta, Kohei; Yamamoto, Miyuki; Lin, Yanling; Hogg, Nathanael; Akiyama, Haruhiko; Behringer, Richard R.; Yamazaki, Yukiko

    2012-01-01

    ABSTRACT Retinoic acid (RA) is a meiosis-inducing factor. Primordial germ cells (PGCs) in the developing ovary are exposed to RA, resulting in entry into meiosis. In contrast, PGCs in the developing testis enter mitotic arrest to differentiate into prospermatogonia. Sertoli cells express CYP26B1, an RA-metabolizing enzyme, providing a simple explanation for why XY PGCs do not initiate meios/is. However, regulation of entry into mitotic arrest is likely more complex. To investigate the mechanisms that regulate male germ cell differentiation, we cultured XX and XY germ cells at 11.5 and 12.5 days postcoitus (dpc) with an RA receptor inhibitor. Expression of Stra8, a meiosis initiation gene, was suppressed in all groups. However, expression of Dnmt3l, a male-specific gene, during embryogenesis was elevated but only in 12.5-dpc XY germ cells. This suggests that inhibiting RA signaling is not sufficient for male germ cell differentiation but that the male gonadal environment also contributes to this pathway. To define the influence of Sertoli cells on male germ cell differentiation, Sertoli cells at 12.5, 15.5, and 18.5 dpc were aggregated with 11.5 dpc PGCs, respectively. After culture, PGCs aggregated with 12.5 dpc Sertoli cells increased Nanos2 and Dnmt3l expression. Furthermore, these PGCs established male-specific methylation imprints of the H19 differentially methylated domains. In contrast, PGCs aggregated with Sertoli cells at late embryonic ages did not commit to the male pathway. These findings suggest that male germ cell differentiation is induced both by inhibition of RA signaling and by molecule(s) production by embryonic age-specific Sertoli cells. PMID:22262692

  8. Stable isotope labelling with amino acids in cell culture for human embryonic stem cell proteomic analysis

    DEFF Research Database (Denmark)

    Harkness, Linda; Prokhorova, Tatyana A; Kassem, Moustapha

    2012-01-01

    The identification and quantitative measurements of proteins in human embryonic stem cells (hESC) is a fast growing interdisciplinary area with an enormous impact on understanding the biology of hESC and the mechanism controlling self-renewal and differentiation. Using a quantitative mass...... spectroscopic method of stable isotope labelling with amino acids during cell culture (SILAC), we are able to analyse differential expression of proteins from different cellular compartments and to identify intracellular signalling pathways involved in self-renewal and differentiation. In this chapter, we...

  9. Derivation of Stromal (Skeletal, Mesenchymal) Stem-like cells from Human Embryonic Stem Cells

    DEFF Research Database (Denmark)

    Mahmood, Amer; Harkness, Linda; Abdallah, Basem

    2012-01-01

    Derivation of bone forming cells (osteoblasts) from human embryonic stem cells (hESC) is a pre-requisite for their use in clinical applications. However, there is no standard protocol for differentiating hESC into osteoblastic cells. The aim of this study was to identify the emergence of a human...... stromal (mesenchymal, skeletal) stem cell (hMSC)-like population, known to be osteoblastic cell precursors and to test their osteoblastic differentiation capacity in ex vivo cultures and in vivo. We cultured hESC in a feeder-free environment using serum replacement and as suspension aggregates (embryoid...... bodies; hEBs). Over a 20 day developmental period, the hEBs demonstrated increasing enrichment for cells expressing hMSC markers: CD29, CD44, CD63, CD56, CD71, CD73, CD105, CD106 and CD166 as revealed by immunohistochemical staining and flow cytometry (FACS) analysis. Ex vivo differentiation of h...

  10. Efficient femtosecond driven SOX 17 delivery into mouse embryonic stem cells: Differentiation study

    CSIR Research Space (South Africa)

    Thobakgale, Setumo L

    2017-01-01

    Full Text Available Efficient femtosecond laser driven SOX 17 delivery into mouse embryonic stem cells: Differentiation study S.L Thobakgale1,2, S.L Manoto1, S. Ombinda-Lemboumba1, M. Maaza2, P. Mthunzi-Kufa1,2* 1Council for Scientific and Industrial Research (CSIR) National... and definitive endoderm in vitro. Development Growth and Differentiation, 50(7), pp.585–593 7. Berrill, A. et al., 2004. Assessment of stem cell markers during long-term culture of mouse embryonic stem cells. Cytotechnology, 44(1-2), pp.77–91. 8. Matthew J...

  11. Derivation and characterization of hepatic progenitor cells from human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Dongxin Zhao

    Full Text Available The derivation of hepatic progenitor cells from human embryonic stem (hES cells is of value both in the study of early human liver organogenesis and in the creation of an unlimited source of donor cells for hepatocyte transplantation therapy. Here, we report for the first time the generation of hepatic progenitor cells derived from hES cells. Hepatic endoderm cells were generated by activating FGF and BMP pathways and were then purified by fluorescence activated cell sorting using a newly identified surface marker, N-cadherin. After co-culture with STO feeder cells, these purified hepatic endoderm cells yielded hepatic progenitor colonies, which possessed the proliferation potential to be cultured for an extended period of more than 100 days. With extensive expansion, they co-expressed the hepatic marker AFP and the biliary lineage marker KRT7 and maintained bipotential differentiation capacity. They were able to differentiate into hepatocyte-like cells, which expressed ALB and AAT, and into cholangiocyte-like cells, which formed duct-like cyst structures, expressed KRT19 and KRT7, and acquired epithelial polarity. In conclusion, this is the first report of the generation of proliferative and bipotential hepatic progenitor cells from hES cells. These hES cell-derived hepatic progenitor cells could be effectively used as an in vitro model for studying the mechanisms of hepatic stem/progenitor cell origin, self-renewal and differentiation.

  12. Efficient femtosecond driven SOX 17 delivery into mouse embryonic stem cells: differentiation studies

    Science.gov (United States)

    Thobakgale, Lebogang; Manoto, Sello Lebohang; Lemboumba, Satuurnin Ombinda; Maaza, Malik; Mthunzi-Kufa, Patience

    2017-02-01

    Embryonic stem cells have great promise in regenerative medicine because of their ability to self-renew and differentiate into various cell types. Delivery of therapeutic genes into cells has already been achieved using of chemical agents and viral vectors with high transfection efficiencies. However, these methods have also been documented as toxic and in the latter case they can cause latent cell infections. In this study we use femtosecond laser pulses to optically deliver genetic material in mouse embryonic stem cells. Femtosecond laser pulses in contrast to the conventional approach, minimises the risk of unwanted side effects because photons are used to create transient pores on the membrane which allow free entry of molecules with no need for delivery agents. Using an Olympus microscope, fluorescence imaging of the samples post irradiation was performed and decreased expression of stage specific embryonic antigen one (SSEA-1) consistent with on-going cellular differentiation was observed. Our results also show that femtosecond laser pulses were effective in delivering SOX 17 plasmid DNA (pSOX17) which resulted in the differentiation of mouse embryonic stem cells into endoderm cells. We thus concluded that laser transfection of stem cells for the purpose of differentiation, holds potential for applications in tissue engineering as a method of generating new cell lines.

  13. High throughput sequencing identifies an imprinted gene, Grb10, associated with the pluripotency state in nuclear transfer embryonic stem cells.

    Science.gov (United States)

    Li, Hui; Gao, Shuai; Huang, Hua; Liu, Wenqiang; Huang, Huanwei; Liu, Xiaoyu; Gao, Yawei; Le, Rongrong; Kou, Xiaochen; Zhao, Yanhong; Kou, Zhaohui; Li, Jia; Wang, Hong; Zhang, Yu; Wang, Hailin; Cai, Tao; Sun, Qingyuan; Gao, Shaorong; Han, Zhiming

    2017-07-18

    Somatic cell nuclear transfer and transcription factor mediated reprogramming are two widely used techniques for somatic cell reprogramming. Both fully reprogrammed nuclear transfer embryonic stem cells and induced pluripotent stem cells hold potential for regenerative medicine, and evaluation of the stem cell pluripotency state is crucial for these applications. Previous reports have shown that the Dlk1-Dio3 region is associated with pluripotency in induced pluripotent stem cells and the incomplete somatic cell reprogramming causes abnormally elevated levels of genomic 5-methylcytosine in induced pluripotent stem cells compared to nuclear transfer embryonic stem cells and embryonic stem cells. In this study, we compared pluripotency associated genes Rian and Gtl2 in the Dlk1-Dio3 region in exactly syngeneic nuclear transfer embryonic stem cells and induced pluripotent stem cells with same genomic insertion. We also assessed 5-methylcytosine and 5-hydroxymethylcytosine levels and performed high-throughput sequencing in these cells. Our results showed that Rian and Gtl2 in the Dlk1-Dio3 region related to pluripotency in induced pluripotent stem cells did not correlate with the genes in nuclear transfer embryonic stem cells, and no significant difference in 5-methylcytosine and 5-hydroxymethylcytosine levels were observed between fully and partially reprogrammed nuclear transfer embryonic stem cells and induced pluripotent stem cells. Through syngeneic comparison, our study identifies for the first time that Grb10 is associated with the pluripotency state in nuclear transfer embryonic stem cells.

  14. Cloning of embryonal stem cell-specific genes: characterization of the transcriptionally controlled gene esg-1.

    Science.gov (United States)

    Bierbaum, P; MacLean-Hunter, S; Ehlert, F; Möröy, T; Müller, R

    1994-01-01

    We have isolated, by differential library screening, eight cDNAs representing genes that are specifically expressed in the embryonal stem cell line IMT-11, when compared to the parietal endoderm-like cell line PYS-2 or to NIH3T3 fibroblasts. One of these genes, embryonal stem cell gene 1 (esg-1), was analyzed in detail. esg-1 mRNA is found at high levels in both IMT-11 and F9 embryonal carcinoma cells and disappears during the differentiation of the stem cells. Furthermore, expression of the gene was found to be extremely low in, or absent from, oocytes and fertilized eggs, but it is strongly induced at the 2-cell stage, reaching maximum levels at the 4-cell stage. In contrast, esg-1 expression is detectable neither in midgestation embryos nor in neonatal tissues. These results strongly suggest that esg-1 is expressed specifically or at least predominantly in embryonal stem cells. Antibodies directed against a glutathione S-transferase-esg-1 fusion product detect a protein of M(r) approximately 14,000 in F9 embryonal carcinoma cells, but not in differentiated cells. Apart from the esg-1 gene, which contains two introns, there are at least seven esg-1-related pseudogenes in the mouse genome that differ from the esg-1 gene by the presence of multiple point mutations, by the lack of intervening sequences, and/or by the presence of a polyadenylated stretch at the 3' end. The esg-1 gene is under stringent transcriptional control in differentiating and differentiated cells, as shown by both nuclear run-on assays and the transient F9 stem cell-specific expression of constructs consisting of esg-1 upstream sequences fused to a luciferase reporter gene.

  15. Gastrin: a distinct fate of neurogenin3 positive progenitor cells in the embryonic pancreas.

    Directory of Open Access Journals (Sweden)

    Yaron Suissa

    Full Text Available Neurogenin3(+ (Ngn3(+ progenitor cells in the developing pancreas give rise to five endocrine cell types secreting insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. Gastrin is a hormone produced primarily by G-cells in the stomach, where it functions to stimulate acid secretion by gastric parietal cells. Gastrin is expressed in the embryonic pancreas and is common in islet cell tumors, but the lineage and regulators of pancreatic gastrin(+ cells are not known. We report that gastrin is abundantly expressed in the embryonic pancreas and disappears soon after birth. Some gastrin(+ cells in the developing pancreas co-express glucagon, ghrelin or pancreatic polypeptide, but many gastrin(+ cells do not express any other islet hormone. Pancreatic gastrin(+ cells express the transcription factors Nkx6.1, Nkx2.2 and low levels of Pdx1, and derive from Ngn3(+ endocrine progenitor cells as shown by genetic lineage tracing. Using mice deficient for key transcription factors we show that gastrin expression depends on Ngn3, Nkx2.2, NeuroD1 and Arx, but not Pax4 or Pax6. Finally, gastrin expression is induced upon differentiation of human embryonic stem cells to pancreatic endocrine cells expressing insulin. Thus, gastrin(+ cells are a distinct endocrine cell type in the pancreas and an alternative fate of Ngn3+ cells.

  16. Gastrin: a distinct fate of neurogenin3 positive progenitor cells in the embryonic pancreas.

    Science.gov (United States)

    Suissa, Yaron; Magenheim, Judith; Stolovich-Rain, Miri; Hija, Ayat; Collombat, Patrick; Mansouri, Ahmed; Sussel, Lori; Sosa-Pineda, Beatriz; McCracken, Kyle; Wells, James M; Heller, R Scott; Dor, Yuval; Glaser, Benjamin

    2013-01-01

    Neurogenin3(+) (Ngn3(+)) progenitor cells in the developing pancreas give rise to five endocrine cell types secreting insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. Gastrin is a hormone produced primarily by G-cells in the stomach, where it functions to stimulate acid secretion by gastric parietal cells. Gastrin is expressed in the embryonic pancreas and is common in islet cell tumors, but the lineage and regulators of pancreatic gastrin(+) cells are not known. We report that gastrin is abundantly expressed in the embryonic pancreas and disappears soon after birth. Some gastrin(+) cells in the developing pancreas co-express glucagon, ghrelin or pancreatic polypeptide, but many gastrin(+) cells do not express any other islet hormone. Pancreatic gastrin(+) cells express the transcription factors Nkx6.1, Nkx2.2 and low levels of Pdx1, and derive from Ngn3(+) endocrine progenitor cells as shown by genetic lineage tracing. Using mice deficient for key transcription factors we show that gastrin expression depends on Ngn3, Nkx2.2, NeuroD1 and Arx, but not Pax4 or Pax6. Finally, gastrin expression is induced upon differentiation of human embryonic stem cells to pancreatic endocrine cells expressing insulin. Thus, gastrin(+) cells are a distinct endocrine cell type in the pancreas and an alternative fate of Ngn3+ cells.

  17. The polycomb group protein Suz12 is required for embryonic stem cell differentiation

    DEFF Research Database (Denmark)

    Pasini, Diego; Bracken, Adrian P; Hansen, Jacob Bo Højberg

    2007-01-01

    results in early lethality of mouse embryos. Here, we demonstrate that Suz12(-/-) mouse embryonic stem (ES) cells can be established and expanded in tissue culture. The Suz12(-/-) ES cells are characterized by global loss of H3K27 trimethylation (H3K27me3) and higher expression levels of differentiation...

  18. In vitro generation of motor neuron precursors from mouse embryonic stem cells using mesoporous nanoparticles

    DEFF Research Database (Denmark)

    Garcia-Bennett, Alfonso E; König, Niclas; Abrahamsson, Ninnie

    2014-01-01

    Aim: Stem cell-derived motor neurons (MNs) are utilized to develop replacement strategies for spinal cord disorders. Differentiation of embryonic stem cells into MN precursors involves factors and their repeated administration. We investigated if delivery of factors loaded into mesoporous...

  19. Global phosphoproteome profiling reveals unanticipated networks responsive to cisplatin treatment of embryonic stem cells

    DEFF Research Database (Denmark)

    Pines, Alex; Kelstrup, Christian D; Vrouwe, Mischa G

    2011-01-01

    (stable isotope labeling by amino acids in cell culture)-labeled murine embryonic stem cells with the anticancer drug cisplatin. Network and pathway analyses indicated that processes related to the DNA damage response and cytoskeleton organization were significantly affected. Although the ATM (ataxia...

  20. Procedures for Derivation and Characterisation of Human Embryonic Stem Cells from Odense, Denmark

    DEFF Research Database (Denmark)

    Harkness, Linda; Kassem, Moustapha

    2012-01-01

    In 1998, a development occurred in stem cell biology with the fi rst report of the derivation of a human embryonic stem cell (hESC) line. Since then a number of techniques have been used to derive and characterise hESCs. Here, we describe the derivation methods used by our laboratory for isolation...

  1. Activin B mediated induction of Pdx1 in human embryonic stem cell derived embryoid bodies

    DEFF Research Database (Denmark)

    Frandsen, Ulrik; Pørneki, Ann Dorte Storm; Floridon, Charlotte

    2007-01-01

    embryonic and fetal pancreas anlage in humans. Pdx1(+) cells are found in cell clusters also expressing Serpina1 and FABP1, suggesting activation of intestinal/liver developmental programs. Moreover, Activin B up-regulates Sonic Hedgehog (Shh) and its target Gli1, which during normal development...

  2. Toward Development of Pluripotent Porcine Stem Cells by Road Mapping Early Embryonic Development

    DEFF Research Database (Denmark)

    Petkov, Stoyan; Freude, Kristine; Mashayekhi-Nezamabadi, Kaveh

    2017-01-01

    The lack in production of bona fide porcine pluripotent stem cells has definitely been hampered by a lack of research into porcine embryo development. Embryonic development in mammals is the extraordinary transition of a single-celled fertilized zygote into a complex fetus, which occurs in the ut...

  3. YAP/TAZ enhance mammalian embryonic neural stem cell characteristics in a Tead-dependent manner

    Energy Technology Data Exchange (ETDEWEB)

    Han, Dasol; Byun, Sung-Hyun; Park, Soojeong; Kim, Juwan; Kim, Inhee; Ha, Soobong; Kwon, Mookwang; Yoon, Keejung, E-mail: keejung@skku.edu

    2015-02-27

    Mammalian brain development is regulated by multiple signaling pathways controlling cell proliferation, migration and differentiation. Here we show that YAP/TAZ enhance embryonic neural stem cell characteristics in a cell autonomous fashion using diverse experimental approaches. Introduction of retroviral vectors expressing YAP or TAZ into the mouse embryonic brain induced cell localization in the ventricular zone (VZ), which is the embryonic neural stem cell niche. This change in cell distribution in the cortical layer is due to the increased stemness of infected cells; YAP-expressing cells were colabeled with Sox2, a neural stem cell marker, and YAP/TAZ increased the frequency and size of neurospheres, indicating enhanced self-renewal- and proliferative ability of neural stem cells. These effects appear to be TEA domain family transcription factor (Tead)–dependent; a Tead binding-defective YAP mutant lost the ability to promote neural stem cell characteristics. Consistently, in utero gene transfer of a constitutively active form of Tead2 (Tead2-VP16) recapitulated all the features of YAP/TAZ overexpression, and dominant negative Tead2-EnR resulted in marked cell exit from the VZ toward outer cortical layers. Taken together, these results indicate that the Tead-dependent YAP/TAZ signaling pathway plays important roles in neural stem cell maintenance by enhancing stemness of neural stem cells during mammalian brain development. - Highlights: • Roles of YAP and Tead in vivo during mammalian brain development are clarified. • Expression of YAP promotes embryonic neural stem cell characteristics in vivo in a cell autonomous fashion. • Enhancement of neural stem cell characteristics by YAP depends on Tead. • Transcriptionally active form of Tead alone can recapitulate the effects of YAP. • Transcriptionally repressive form of Tead severely reduces stem cell characteristics.

  4. Evaluation of biological effects of intermediate frequency magnetic field on differentiation of embryonic stem cell

    Directory of Open Access Journals (Sweden)

    Sachiko Yoshie

    2016-01-01

    Full Text Available The embryotoxic effect of intermediate frequency (IF magnetic field (MF was evaluated using murine embryonic stem (ES cells and fibroblast cells based on the embryonic stem cell test (EST. The cells were exposed to 21 kHz IF–MF up to magnetic flux density of 3.9 mT during the cell proliferation process (7 days or the cell differentiation process (10 days during which an embryonic body differentiated into myocardial cells. As a result, there was no significant difference in the cell proliferation between sham- and IF–MF-exposed cells for both ES and fibroblast cells. Similarly, the ratio of the number of ES-derived cell aggregates differentiated to myocardial cells to total number of cell aggregates was not changed by IF–MF exposure. In addition, the expressions of a cardiomyocytes-specific gene, Myl2, and an early developmental gene, Hba-x, in the exposed cell aggregate were not altered. Since the magnetic flux density adopted in this study is much higher than that generated by an inverter of the electrical railway, an induction heating (IH cooktop, etc. in our daily lives, these results suggested that IF–MF in which the public is exposed to in general living environment would not have embryotoxic effect.

  5. DNA damage responses in human induced pluripotent stem cells and embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Olga Momcilovic

    2010-10-01

    Full Text Available Induced pluripotent stem (iPS cells have the capability to undergo self-renewal and differentiation into all somatic cell types. Since they can be produced through somatic cell reprogramming, which uses a defined set of transcription factors, iPS cells represent important sources of patient-specific cells for clinical applications. However, before these cells can be used in therapeutic designs, it is essential to understand their genetic stability.Here, we describe DNA damage responses in human iPS cells. We observe hypersensitivity to DNA damaging agents resulting in rapid induction of apoptosis after γ-irradiation. Expression of pluripotency factors does not appear to be diminished after irradiation in iPS cells. Following irradiation, iPS cells activate checkpoint signaling, evidenced by phosphorylation of ATM, NBS1, CHEK2, and TP53, localization of ATM to the double strand breaks (DSB, and localization of TP53 to the nucleus of NANOG-positive cells. We demonstrate that iPS cells temporary arrest cell cycle progression in the G(2 phase of the cell cycle, displaying a lack of the G(1/S cell cycle arrest similar to human embryonic stem (ES cells. Furthermore, both cell types remove DSB within six hours of γ-irradiation, form RAD51 foci and exhibit sister chromatid exchanges suggesting homologous recombination repair. Finally, we report elevated expression of genes involved in DNA damage signaling, checkpoint function, and repair of various types of DNA lesions in ES and iPS cells relative to their differentiated counterparts.High degrees of similarity in DNA damage responses between ES and iPS cells were found. Even though reprogramming did not alter checkpoint signaling following DNA damage, dramatic changes in cell cycle structure, including a high percentage of cells in the S phase, increased radiosensitivity and loss of DNA damage-induced G(1/S cell cycle arrest, were observed in stem cells generated by induced pluripotency.

  6. Human embryonic stem cell-derived neuronal cells form spontaneously active neuronal networks in vitro.

    Science.gov (United States)

    Heikkilä, Teemu J; Ylä-Outinen, Laura; Tanskanen, Jarno M A; Lappalainen, Riikka S; Skottman, Heli; Suuronen, Riitta; Mikkonen, Jarno E; Hyttinen, Jari A K; Narkilahti, Susanna

    2009-07-01

    The production of functional human embryonic stem cell (hESC)-derived neuronal cells is critical for the application of hESCs in treating neurodegenerative disorders. To study the potential functionality of hESC-derived neurons, we cultured and monitored the development of hESC-derived neuronal networks on microelectrode arrays. Immunocytochemical studies revealed that these networks were positive for the neuronal marker proteins beta-tubulin(III) and microtubule-associated protein 2 (MAP-2). The hESC-derived neuronal networks were spontaneously active and exhibited a multitude of electrical impulse firing patterns. Synchronous bursts of electrical activity similar to those reported for hippocampal neurons and rodent embryonic stem cell-derived neuronal networks were recorded from the differentiated cultures until up to 4 months. The dependence of the observed neuronal network activity on sodium ion channels was examined using tetrodotoxin (TTX). Antagonists for the glutamate receptors NMDA [D(-)-2-amino-5-phosphonopentanoic acid] and AMPA/kainate [6-cyano-7-nitroquinoxaline-2,3-dione], and for GABAA receptors [(-)-bicuculline methiodide] modulated the spontaneous electrical activity, indicating that pharmacologically susceptible neuronal networks with functional synapses had been generated. The findings indicate that hESC-derived neuronal cells can generate spontaneously active networks with synchronous communication in vitro, and are therefore suitable for use in developmental and drug screening studies, as well as for regenerative medicine.

  7. Asynchronous replication and autosome-pair non-equivalence in human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Devkanya Dutta

    Full Text Available A number of mammalian genes exhibit the unusual properties of random monoallelic expression and random asynchronous replication. Such exceptional genes include genes subject to X inactivation and autosomal genes including odorant receptors, immunoglobulins, interleukins, pheromone receptors, and p120 catenin. In differentiated cells, random asynchronous replication of interspersed autosomal genes is coordinated at the whole chromosome level, indicative of chromosome-pair non-equivalence. Here we have investigated the replication pattern of the random asynchronously replicating genes in undifferentiated human embryonic stem cells, using fluorescence in situ hybridization based assay. We show that allele-specific replication of X-linked genes and random monoallelic autosomal genes occur in human embryonic stem cells. The direction of replication is coordinated at the whole chromosome level and can cross the centromere, indicating the existence of autosome-pair non-equivalence in human embryonic stem cells. These results suggest that epigenetic mechanism(s that randomly distinguish between two parental alleles are emerging in the cells of the inner cell mass, the source of human embryonic stem cells.

  8. Alginate Encapsulation Parameters Influence the Differentiation of Microencapsulated Embryonic Stem Cell Aggregates

    OpenAIRE

    Wilson, Jenna L.; Najia, Mohamad Ali; Saeed, Rabbia; McDevitt, Todd C.

    2013-01-01

    Pluripotent embryonic stem cells (ESCs) have tremendous potential as tools for regenerative medicine and drug discovery, yet the lack of processes to manufacture viable and homogenous cell populations of sufficient numbers limits the clinical translation of current and future cell therapies. Microencapsulation of ESCs within microbeads can shield cells from hydrodynamic shear forces found in bioreactor environments while allowing for sufficient diffusion of nutrients and oxygen through the en...

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

    OpenAIRE

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

    2011-01-01

    The successful implementation of human embryonic stem cells (hESCs)-based technologies requires the production of relevant numbers of well-characterized cells and their efficient long-term storage. In this study, cells were microencapsulated in alginate to develop an integrated bioprocess for expansion and cryopreservation of pluripotent hESCs. Different three-dimensional (3D) culture strategies were evaluated and compared, specifically, microencapsulation of hESCs as: i) single cells, ii) ag...

  10. Differentiation of mouse embryonic stem cells into a defined neuronal lineage.

    Science.gov (United States)

    Bibel, Miriam; Richter, Jens; Schrenk, Katrin; Tucker, Kerry Lee; Staiger, Volker; Korte, Martin; Goetz, Magdalena; Barde, Yves-Alain

    2004-09-01

    Although it has long been known that cultured embryonic stem cells can generate neurons, the lineage relationships with their immediate precursors remain unclear. We report here that selection of highly proliferative stem cells followed by treatment with retinoic acid generated essentially pure precursors that markers identified as Pax-6-positive radial glial cells. As they do in vivo, these cells went on to generate neurons with remarkably uniform biochemical and electrophysiological characteristics.

  11. Effect of passage number on electrophoretic mobility distributions of cultured human embryonic kidney cells

    Science.gov (United States)

    Kunze, M. E.

    1985-01-01

    A systematic investigation was undertaken to characterize population shifts that occur in cultured human embryonic kidney cells as a function of passage number in vitro after original explantation. This approach to cell population shift analysis follows the suggestion of Mehreshi, Klein and Revesz that perturbed cell populations can be characterized by electrophoretic mobility distributions if they contain subpopulations with different electrophoretic mobilities. It was shown that this is the case with early passage cultured human embryo cells.

  12. Generation of murine sympathoadrenergic progenitor-like cells from embryonic stem cells and postnatal adrenal glands.

    Science.gov (United States)

    Saxena, Shobhit; Wahl, Joachim; Huber-Lang, Markus S; Stadel, Dominic; Braubach, Peter; Debatin, Klaus-Michael; Beltinger, Christian

    2013-01-01

    Sympathoadrenergic progenitor cells (SAPs) of the peripheral nervous system (PNS) are important for normal development of the sympathetic PNS and for the genesis of neuroblastoma, the most common and often lethal extracranial solid tumor in childhood. However, it remains difficult to isolate sufficient numbers of SAPs for investigations. We therefore set out to improve generation of SAPs by using two complementary approaches, differentiation from murine embryonic stem cells (ESCs) and isolation from postnatal murine adrenal glands. We provide evidence that selecting for GD2 expression enriches for ESC-derived SAP-like cells and that proliferating SAP-like cells can be isolated from postnatal adrenal glands of mice. These advances may facilitate investigations about the development and malignant transformation of the sympathetic PNS.

  13. Oct-4 controls cell-cycle progression of embryonic stem cells

    Science.gov (United States)

    Lee, Jungwoon; Go, Yeorim; Kang, Inyoung; Han, Yong-Mahn; Kim, Jungho

    2009-01-01

    Mouse and human ES (embryonic stem) cells display unusual proliferative properties and can produce pluripotent stem cells indefinitely. Both processes might be important for maintaining the ‘stemness’ of ES cells; however, little is known about how the cell-cycle fate is regulated in ES cells. Oct-4, a master switch of pluripotency, plays an important role in maintaining the pluripotent state of ES cells and may prevent the expression of genes activated during differentiation. Using ZHBTc4 ES cells, we have investigated the effect of Oct-4 on ES cell-cycle control, and we found that Oct-4 down-regulation in ES cells inhibits proliferation by blocking cell-cycle progression in G0/G1. Deletion analysis of the functional domains of Oct-4 indicates that the overall integrity of the Oct-4 functional domains is important for the stimulation of S-phase entry. We also show in the present study that the p21 gene is a target for Oct-4 repression. Furthermore, p21 protein levels were repressed by Oct-4 and were induced by the down-regulation of Oct-4 in ZHBTc4 ES cells. Therefore the down-regulation of p21 by Oct-4 may contribute to the maintenance of ES cell proliferation. PMID:19968627

  14. Neuroblastoma cells injected into experimental mature teratoma reveal a tropism for embryonic loose mesenchyme.

    Science.gov (United States)

    Jamil, S; Cedervall, J; Hultman, I; Ali, R; Margaryan, N V; Rasmuson, A; Johnsen, J I; Sveinbjörnsson, B; Dalianis, T; Kanter, L; Orrego, A; Strizzi, L; Hendrix, M J C; Sandstedt, B; Kogner, P; Ahrlund-Richter, L

    2013-09-01

    Embryonic neural tumors are responsible for a disproportionate number of cancer deaths in children. Although dramatic improvements in survival for pediatric malignancy has been achieved in previous years advancements seem to be slowing down. For the development of new enhanced therapy and an increased understanding of the disease, pre-clinical models better capturing the neoplastic niche are essential. Tumors of early childhood present in this respect a particular challenge. Here, we explore how components of the embryonic process in stem‑cell induced mature teratoma can function as an experimental in vivo microenvironment instigating the growth of injected childhood neuroblastoma (NB) cell lines. Three human NB cell lines, IMR-32, Kelly and SK-N-BE(2), were injected into mature pluripotent stem cell‑induced teratoma (PSCT) and compared to xenografts of the same cell lines. Proliferative NB cells from all lines were readily detected in both models with a typical histology of a poorly differentiated NB tumor with a variable amount of fibrovascular stroma. Uniquely in the PSCT microenvironment, NB cells were found integrated in a non‑random fashion. Neuroblastoma cells were never observed in areas with well-differentiated somatic tissue i.e. bone, muscle, gut or areas of other easily identifiable tissue types. Instead, the three cell lines all showed initial growth exclusively occurring in the embryonic loose mesenchymal stroma, resulting in a histology recapitulating NB native presentation in vivo. Whether this reflects the 'open' nature of loose mesenchyme more easily giving space to new cells compared to other more dense tissues, the rigidity of matrix providing physical cues modulating NB characteristics, or if embryonic loose mesenchyme may supply developmental cues that attracted or promoted the integration of NB, remains to be tested. We tentatively hypothesize that mature PSCT provide an embryonic niche well suited for in vivo studies on NB.

  15. High glucose suppresses embryonic stem cell differentiation into cardiomyocytes : High glucose inhibits ES cell cardiogenesis.

    Science.gov (United States)

    Yang, Penghua; Chen, Xi; Kaushal, Sunjay; Reece, E Albert; Yang, Peixin

    2016-12-09

    Babies born to mothers with pregestational diabetes have a high risk for congenital heart defects (CHD). Embryonic stem cells (ESCs) are excellent in vitro models for studying the effect of high glucose on cardiac lineage specification because ESCs can be differentiated into cardiomyocytes. ESC maintenance and differentiation are currently performed under high glucose conditions, whose adverse effects have never been clarified. We investigated the effect of high glucose on cardiomyocyte differentiation from a well-characterized ESC line, E14, derived from mouse blastocysts. E14 cells maintained under high glucose (25 mM) failed to generate any beating cardiomyocytes using the hanging-drop embryonic body method. We created a glucose-responsive E14 cell line (GR-E14) through a graduated low glucose adaptation. The expression of stem cell markers was similar in the parent E14 cells and the GR-E14 cells. Glucose transporter 2 gene was increased in GR-E14 cells. When GR-E14 cells were differentiated into cardiomyocytes under low (5 mM) or high (25 mM) glucose conditions, high glucose significantly delayed the appearance and reduced the number of TNNT2 (Troponin T Type 2)-positive contracting cardiomyocytes. High glucose suppressed the expression of precardiac mesoderm markers, cardiac transcription factors, mature cardiomyocyte markers, and potassium channel proteins. High glucose impaired the functionality of ESC-derived cardiomyocytes by suppressing the frequencies of Ca 2+ wave and contraction. Our findings suggest that high glucose inhibits ESC cardiogenesis by suppressing key developmental genes essential for the cardiac program.

  16. Maturation of spinal motor neurons derived from human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Tomonori Takazawa

    Full Text Available Our understanding of motor neuron biology in humans is derived mainly from investigation of human postmortem tissue and more indirectly from live animal models such as rodents. Thus generation of motor neurons from human embryonic stem cells and human induced pluripotent stem cells is an important new approach to model motor neuron function. To be useful models of human motor neuron function, cells generated in vitro should develop mature properties that are the hallmarks of motor neurons in vivo such as elaborated neuronal processes and mature electrophysiological characteristics. Here we have investigated changes in morphological and electrophysiological properties associated with maturation of neurons differentiated from human embryonic stem cells expressing GFP driven by a motor neuron specific reporter (Hb9::GFP in culture. We observed maturation in cellular morphology seen as more complex neurite outgrowth and increased soma area over time. Electrophysiological changes included decreasing input resistance and increasing action potential firing frequency over 13 days in vitro. Furthermore, these human embryonic stem cell derived motor neurons acquired two physiological characteristics that are thought to underpin motor neuron integrated function in motor circuits; spike frequency adaptation and rebound action potential firing. These findings show that human embryonic stem cell derived motor neurons develop functional characteristics typical of spinal motor neurons in vivo and suggest that they are a relevant and useful platform for studying motor neuron development and function and for modeling motor neuron diseases.

  17. Nicotine induces mitochondrial fission through mitofusin degradation in human multipotent embryonic carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Hirata, Naoya; Yamada, Shigeru [Division of Pharmacology, National Institute of Health Sciences (Japan); Asanagi, Miki [Division of Pharmacology, National Institute of Health Sciences (Japan); Faculty of Engineering, Department of Materials Science and Engineering, Yokohama National University (Japan); Sekino, Yuko [Division of Pharmacology, National Institute of Health Sciences (Japan); Kanda, Yasunari, E-mail: kanda@nihs.go.jp [Division of Pharmacology, National Institute of Health Sciences (Japan)

    2016-02-05

    Nicotine is considered to contribute to the health risks associated with cigarette smoking. Nicotine exerts its cellular functions by acting on nicotinic acetylcholine receptors (nAChRs), and adversely affects normal embryonic development. However, nicotine toxicity has not been elucidated in human embryonic stage. In the present study, we examined the cytotoxic effects of nicotine in human multipotent embryonal carcinoma cell line NT2/D1. We found that exposure to 10 μM nicotine decreased intracellular ATP levels and inhibited proliferation of NT2/D1 cells. Because nicotine suppressed energy production, which is a critical mitochondrial function, we further assessed the effects of nicotine on mitochondrial dynamics. Staining with MitoTracker revealed that 10 μM nicotine induced mitochondrial fragmentation. The levels of the mitochondrial fusion proteins, mitofusins 1 and 2, were also reduced in cells exposed to nicotine. These nicotine effects were blocked by treatment with mecamylamine, a nonselective nAChR antagonist. These data suggest that nicotine degrades mitofusin in NT2/D1 cells and thus induces mitochondrial dysfunction and cell growth inhibition in a nAChR-dependent manner. Thus, mitochondrial function in embryonic cells could be used to assess the developmental toxicity of chemicals.

  18. Maturation of Spinal Motor Neurons Derived from Human Embryonic Stem Cells

    Science.gov (United States)

    Takazawa, Tomonori; Croft, Gist F.; Amoroso, Mackenzie W.; Studer, Lorenz; Wichterle, Hynek; MacDermott, Amy B.

    2012-01-01

    Our understanding of motor neuron biology in humans is derived mainly from investigation of human postmortem tissue and more indirectly from live animal models such as rodents. Thus generation of motor neurons from human embryonic stem cells and human induced pluripotent stem cells is an important new approach to model motor neuron function. To be useful models of human motor neuron function, cells generated in vitro should develop mature properties that are the hallmarks of motor neurons in vivo such as elaborated neuronal processes and mature electrophysiological characteristics. Here we have investigated changes in morphological and electrophysiological properties associated with maturation of neurons differentiated from human embryonic stem cells expressing GFP driven by a motor neuron specific reporter (Hb9::GFP) in culture. We observed maturation in cellular morphology seen as more complex neurite outgrowth and increased soma area over time. Electrophysiological changes included decreasing input resistance and increasing action potential firing frequency over 13 days in vitro. Furthermore, these human embryonic stem cell derived motor neurons acquired two physiological characteristics that are thought to underpin motor neuron integrated function in motor circuits; spike frequency adaptation and rebound action potential firing. These findings show that human embryonic stem cell derived motor neurons develop functional characteristics typical of spinal motor neurons in vivo and suggest that they are a relevant and useful platform for studying motor neuron development and function and for modeling motor neuron diseases. PMID:22802953

  19. Which bank? A guardian model for regulation of embryonic stem cell research in Australia.

    Science.gov (United States)

    McLennan, A

    2007-08-01

    In late 2005 the Legislation Review: Prohibition of Human Cloning Act 2002 (Cth) and the Research Involving Human Embryos Act 2002 (Cth) recommended the establishment of an Australian stem cell bank. This article aims to address a lack of discussion of issues surrounding stem cell banking by suggesting possible answers to the questions of whether Australia should establish a stem cell bank and what its underlying philosophy and functions should be. Answers are developed through an analysis of regulatory, scientific and intellectual property issues relating to embryonic stem cell research in the United Kingdom, United States and Australia. This includes a detailed analysis of the United Kingdom Stem Cell Bank. It is argued that a "guardian" model stem cell bank should be established in Australia. This bank would aim to promote the maximum public benefit from human embryonic stem cell research by providing careful regulatory oversight and addressing ethical issues, while also facilitating research by addressing practical scientific concerns and intellectual property issues.

  20. High expression of hTERT and stemness genes in BORIS/CTCFL positive cells isolated from embryonic cancer cells.

    Directory of Open Access Journals (Sweden)

    Loredana Alberti

    Full Text Available BORIS/CTCFL is a member of cancer testis antigen family normally expressed in germ cells. In tumors, it is aberrantly expressed although its functions are not completely well-defined. To better understand the functions of BORIS in cancer, we selected the embryonic cancer cells as a model. Using a molecular beacon, which specifically targets BORIS mRNA, we demonstrated that BORIS positive cells are a small subpopulation of tumor cells (3-5% of total. The BORIS-positive cells isolated using BORIS-molecular beacon, expressed higher telomerase hTERT, stem cell (NANOG, OCT4, SOX2 and cancer stem cell marker genes (CD44 and ALDH1 compared to the BORIS-negative tumor cells. In order to define the functional role of BORIS, stable BORIS-depleted embryonic cancer cells were generated. BORIS silencing strongly down-regulated the expression of hTERT, stem cell and cancer stem cell marker genes. Moreover, the BORIS knockdown increased cellular senescence in embryonic cancer cells, revealing a putative role of BORIS in the senescence biological program. Our data indicate an association of BORIS expressing cells subpopulation with the expression of stemness genes, highlighting the critical role played by BORIS in embryonic neoplastic disease.

  1. Effects of Feeder Cell Types on Culture of Mouse Embryonic Stem Cell In Vitro.

    Science.gov (United States)

    Park, Yun-Gwi; Lee, Seung-Eun; Kim, Eun-Young; Hyun, Hyuk; Shin, Min-Young; Son, Yeo-Jin; Kim, Su-Young; Park, Se-Pill

    2015-09-01

    The suitable feeder cell layer is important for culture of embryonic stem (ES) cells. In this study, we investigated the effect of two kinds of the feeder cell, MEF cells and STO cells, layer to mouse ES (mES) cell culture for maintenance of stemness. We compare the colony formations, alkaline phosphatase (AP) activities, expression of pluripotency marker genes and proteins of D3 cell colonies cultured on MEF feeder cell layer (D3/MEF) or STO cell layers (D3/STO) compared to feeder free condition (D3/-) as a control group. Although there were no differences to colony formations and AP activities, interestingly, the transcripts level of pluripotency marker genes, Pou5f1 and Nanog were highly expressed in D3/MEF (79 and 93) than D3/STO (61and 77) or D3/- (65 and 81). Also, pluripotency marker proteins, NANOG and SOX-2, were more synthesized in D3/MEF (72.8±7.69 and 81.2±3.56) than D3/STO (32.0±4.30 and 56.0±4.90) or D3/- (55.0±4.64 and 62.0±6.20). These results suggest that MEF feeder cell layer is more suitable to mES cell culture.

  2. Identification of human embryonic progenitor cell targeting peptides using phage display.

    Directory of Open Access Journals (Sweden)

    Paola A Bignone

    Full Text Available Human pluripotent stem (hPS cells are capable of differentiation into derivatives of all three primary embryonic germ layers and can self-renew indefinitely. They therefore offer a potentially scalable source of replacement cells to treat a variety of degenerative diseases. The ability to reprogram adult cells to induced pluripotent stem (iPS cells has now enabled the possibility of patient-specific hPS cells as a source of cells for disease modeling, drug discovery, and potentially, cell replacement therapies. While reprogramming technology has dramatically increased the availability of normal and diseased hPS cell lines for basic research, a major bottleneck is the critical unmet need for more efficient methods of deriving well-defined cell populations from hPS cells. Phage display is a powerful method for selecting affinity ligands that could be used for identifying and potentially purifying a variety of cell types derived from hPS cells. However, identification of specific progenitor cell-binding peptides using phage display may be hindered by the large cellular heterogeneity present in differentiating hPS cell populations. We therefore tested the hypothesis that peptides selected for their ability to bind a clonal cell line derived from hPS cells would bind early progenitor cell types emerging from differentiating hPS cells. The human embryonic stem (hES cell-derived embryonic progenitor cell line, W10, was used and cell-targeting peptides were identified. Competition studies demonstrated specificity of peptide binding to the target cell surface. Efficient peptide targeted cell labeling was accomplished using multivalent peptide-quantum dot complexes as detected by fluorescence microscopy and flow cytometry. The cell-binding peptides were selective for differentiated hPS cells, had little or no binding on pluripotent cells, but preferential binding to certain embryonic progenitor cell lines and early endodermal hPS cell derivatives. Taken

  3. Plasma treatment of biomaterials to direct the differentiation of embryonic stem cells

    Science.gov (United States)

    Hanley, Erik

    In this work, we explore how embryonic stem (ES) cell differentiation patterns are affected by surface interactions with plasma-processed materials. We hypothesize that mouse embryonic stem-cell exposure to certain plasma-polymerized tetraglyme surfaces will direct their differentiation into endothelial cells. R1 mouse embryonic stem (ES) cells were plated on surfaces onto which tetraglyme was deposited by plasma polymerization. In addition, tissue-treated polystyrene and control glass cover slips were also examined. Some samples were fixed three days after plating and immunofluorescence stained with platelet endothelial-cell adhesion molecule, while the others were fixed seven days after plating and immunofluorescence stained with von Willebrand Factor. Positive results seen by ES cell derivatives precociously expressing the vWF and PECAM genetic markers on the plasma-polymerized tetraglyme treated surfaces suggest that the plasma-polymerized surfaces direct differentiation of ES cells into endothelial cells. Research goals of this dissertation include: characterization of the material properties of the plasma-polymerized tetraglyme surfaces that induce directed differentiation of ES cells into endothelial cells, optimization of the plasma-polymerization process to maximize the number of endothelial cells derived from R1 ES cells, and biological experimentation to characterize properties of the mechanism of directed differentiation. A potential application of this work is in the design and construction of an artificial blood vessel. Current small-scale arterial substitutes have proved inadequate because of thrombogenicity and infection. Moreover, the lower blood flow velocities of smaller vessels pose a different set of design criteria and introduce new problems not encountered in large arterial substitutes. By utilizing a tissue engineering approach that incorporates embryonic stem cell-derived endothelial cells, the longevity of the prosthesis can be ensured.

  4. Assessment of stem cell markers during long-term culture of mouse embryonic stem cells.

    Science.gov (United States)

    Berrill, A; Tan, H L; Wuang, S C; Fong, W J; Choo, Andre B H; Oh, Steve K W

    2004-01-01

    Embryonic stem (ES) cells have been in the fore front of scientific literature lately as having the potential for regeneration of many tissue types. Two important issues that need to be addressed are the culture conditions for maintaining ES cells and the accuracy of ES cell markers in monitoring the undifferentiated state. Leukaemia inhibitory factor (LIF) is routinely used to sustain mouse ES cells (mES) in a pluripotent fashion. In this paper, we assessed three markers during long-term maintenance of ES cells with various concentrations of LIF to see if decreasing concentration would lead to changes in marker expressions and growth behavior. Common markers of pluripotency such as alkaline phosphatase enzyme activity (ALP), surface staining for stage specific embryonic antigen 1 (SSEA-1), Oct-4 transcription factor, cell doubling time, as well as visual observations of cell morphology were analyzed during long-term maintenance of mES cells with LIF concentrations ranging from 0 to 500 pM. The morphology of the cells at LIF concentrations of 0 25 pM changed from being tight clusters to more flattened shapes while cells in 50-500 pM retained the clustered shape but growth rates remained essentially identical at between 10 and 16 h. ES cells at all concentrations of LIF continued expressing ALP, SSEA-1 and Oct-4 markers over a period of 6 weeks, which indicate that mES cells are capable of either producing autocrine LIF or are able to proliferate at very low levels of LIF. Pluripotency markers such as Oct-4 and SSEA-1 are only moderately reduced after 5-6 weeks. Oct-4 mRNA expression levels were partially diminished in LIF free conditions only at weeks 5 and 6 compared to controls with LIF at 500 pM. Changes in morphology of cells by visual observation seemed to be a faster indication of the onset of differentiation in mES cells, although other reliable means also include decreased levels of Oct-4, SSEA-1 and ALP markers. It is preferable to maintain long

  5. Poly(trimethylene carbonate) as an elastic biodegradable film for human embryonic stem cell-derived retinal pigment epithelial cells

    NARCIS (Netherlands)

    Sorkio, A.; Haimi, Suvi; Verdoold, Vincent; Juuti-Uusitalo, K.; Grijpma, Dirk W.; Skottman, H.

    2017-01-01

    Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cell therapies show tremendous potential for the treatment of retinal degenerative diseases. A tissue engineering approach, where cells are delivered to the subretinal space on a biodegradable carrier as a sheet, shows great

  6. Skeletogenic phenotype of human Marfan embryonic stem cells faithfully phenocopied by patient-specific induced-pluripotent stem cells

    Science.gov (United States)

    Quarto, Natalina; Leonard, Brian; Li, Shuli; Marchand, Melanie; Anderson, Erica; Behr, Barry; Francke, Uta; Reijo-Pera, Renee; Chiao, Eric; Longaker, Michael T.

    2012-01-01

    Marfan syndrome (MFS) is a heritable connective tissue disorder caused by mutations in the gene coding for FIBRILLIN-1 (FBN1), an extracellular matrix protein. MFS is inherited as an autosomal dominant trait and displays major manifestations in the ocular, skeletal, and cardiovascular systems. Here we report molecular and phenotypic profiles of skeletogenesis in tissues differentiated from human embryonic stem cells and induced pluripotent stem cells that carry a heritable mutation in FBN1. We demonstrate that, as a biological consequence of the activation of TGF-β signaling, osteogenic differentiation of embryonic stem cells with a FBN1 mutation is inhibited; osteogenesis is rescued by inhibition of TGF-β signaling. In contrast, chondrogenesis is not perturbated and occurs in a TGF-β cell-autonomous fashion. Importantly, skeletal phenotypes observed in human embryonic stem cells carrying the monogenic FBN1 mutation (MFS cells) are faithfully phenocopied by cells differentiated from induced pluripotent-stem cells derived independently from MFS patient fibroblasts. Results indicate a unique phenotype uncovered by examination of mutant pluripotent stem cells and further demonstrate the faithful alignment of phenotypes in differentiated cells obtained from both human embryonic stem cells and induced pluripotent-stem cells, providing complementary and powerful tools to gain further insights into human molecular pathogenesis, especially of MFS. PMID:22178754

  7. Alginate encapsulation technology supports embryonic stem cells differentiation into insulin-producing cells.

    Science.gov (United States)

    Wang, Nan; Adams, Gary; Buttery, Lee; Falcone, Franco H; Stolnik, Snow

    2009-12-01

    This work investigates an application of the alginate encapsulation technology to the differentiation of embryonic stem (ES) cells into insulin-producing cells. It shows that the ES cells can efficiently be encapsulated within the alginate beads, retaining a high level of cell viability. The alginate encapsulation achieves approximately 10-fold increase in the cell density in the culture, in comparison to the two-dimensional conditions, opening a potential benefit of the technology in large-scale cell culture applications. Manipulations of encapsulation conditions, particularly of the initial alginate concentration, allow the control over both the diffusion of molecules into the alginate matrix (e.g. differentiation factors) as well as control over the matrix porosity/flexibility to permit the proliferation and growth of encapsulated ES aggregates within the bead. Post-differentiation analysis confirms the presence of insulin-positive cells, as judged from immunostaining, insulin ELISA and RT-PCR analysis. The functionality of the encapsulated and differentiated cells was confirmed by their insulin production capability, whereby on glucose challenge the insulin production by the cells differentiated within alginate beads was found to be statistically significantly higher than for the cells from conventional two-dimensional differentiation system.

  8. Restoration of heart functions using human embryonic stem cells derived heart muscle cells.

    Science.gov (United States)

    Gepstein, Lior; Kehat, Izhak

    2005-02-01

    Extract: Recent advances in molecular and cellular biology and specifically in the areas of stem cell biology and tissue engineering have paved the way for the development of a new field in biomedicine, regenerative medicine. This exciting approach seeks to develop new biological solutions, using the mobilization of endogenous stem cells or delivery of exogenous cells to replace or modify the function of diseased, absent, or malfunctioning tissue. The adult heart represents an attractive candidate for these emerging technologies, since adult cardiomyocytes have limited regenerative capacity. Thus, any significant heart cell loss or dysfunction, such as occurs during heart attack, is mostly irreversible and may lead to the development of progressive heart failure, one of the leading causes of world-wide morbidity and mortality. Similarly, dysfunction of the specialized electrical conduction system within the heart may result in inefficient rhythm initiation or impulse conduction, leading to significant slowing of the heart rate, usually requiring the implantation of a permanent electronic pacemaker. Replacement of the dysfunctional myocardium (heart muscle) by implantation of external heart muscle cells is emerging as a novel paradigm for restoration of the myocardial electromechanical properties, but has been significantly hampered by the paucity of cell sources for human heart cells and by the relatively limited evidence for functional integration between grafted and host cells. The recently described human embryonic stem cell (hESC) lines may provide a possible solution for the aforementioned cell sourcing problem.

  9. EXAMINATION OF THE GERM CELL CHIMERA FORMING POTENTIAL OF MOUSE EMBRYONIC STEM CELLS

    Directory of Open Access Journals (Sweden)

    CÂRSTEA V. B

    2007-01-01

    Full Text Available The aim of this study was to examine the factors, which influence the chimeraforming potential of mouse embryonic stem cells (ES cells. In our work, we examinethe chimera producing ability of R1 and R1/E mouse ES cell lines. We found that thepassage number affects chimera-forming capability of the ES cells. With theincreasing of the passage number, it could be getting less chimera animal, and onlythe R1/E ES cell line derived cells could contribute to the germ cells. At first, wecompared the marker of pluripotency using immunostaining and RT PCR, but wecould not find any difference between the R1 and R1/E cell in this way. Atchromosome analysis, we found, that the number of aneuploid cells, in R1 ES cellline, dramatically increased after 10 passages. We thought that the reason is thatduring the cell division Y chromosome could not arrange correctly between the twonewly derived progeny cells. To prove our conception, we made X and YchromosomeFISH analyses. We found, that the aneuploid R1 and R1/E ES cellscontain only one X and one Y chromosome, so not the loss of Y chromosome causethe problem at the germ cell formation. At last, we made the karyotypeanalysis of R1 and R1/E ES cells at different passages. The karyotype analysisdemonstrated that in the case of R1 ES cell line, the 41 and 42-chromosomecontaining cells hold trisomy. With the increasing of the passages number, thenumber of trisomy containing aneuploid cells increased. The aneuploid ES cells cancontribute to the different tissuses of chimera animals, but cannot form viable germcells.

  10. Prospects and Ethical Concerns of Embryonic Stem Cells Research-A Review

    Directory of Open Access Journals (Sweden)

    Jayanti Tokas and P. D. Mathur

    2011-12-01

    Full Text Available Stem cell research has appeared as a silver lining of hope over the dark cloud of some untreatable diseases like cancer and certain neurological disorders. Embryonic stem cells, the tabula rasa, holds much promise in this regard owing to its totipotency, howbeit, it has whirled a severe tempest all over the world on the point of humanity. The present review article includes the chronology of stem cell research with special reference to the techniques that were evolved in due course of research, the controversy over the application of embryonic stem cells for therapeutics and present status of stem cell research under Indian context. India is being increasingly alluring the foreign companies to invest in this project since a huge prospect in stem cell marketing business is foreseen in this country. [Vet. World 2011; 4(6.000: 281-286

  11. Undifferentiated embryonic cell transcription factor 1 regulates ESC chromatin organization and gene expression

    DEFF Research Database (Denmark)

    Kooistra, Susanne M; van den Boom, Vincent; Thummer, Rajkumar P

    2010-01-01

    Previous reports showed that embryonic stem (ES) cells contain hyperdynamic and globally transcribed chromatin-properties that are important for ES cell pluripotency and differentiation. Here, we demonstrate a role for undifferentiated embryonic cell transcription factor 1 (UTF1) in regulating ES...... cell chromatin structure. Using chromatin immunoprecipitation-on-chip analysis, we identified >1,700 UTF1 target genes that significantly overlap with previously identified Nanog, Oct4, Klf-4, c-Myc, and Rex1 targets. Gene expression profiling showed that UTF1 knock down results in increased expression...... of a large set of genes, including a significant number of UTF1 targets. UTF1 knock down (KD) ES cells are, irrespective of the increased expression of several self-renewal genes, Leukemia inhibitory factor (LIF) dependent. However, UTF1 KD ES cells are perturbed in their differentiation in response...

  12. Reconstitution of mammary epithelial morphogenesis by murine embryonic stem cells undergoing hematopoietic stem cell differentiation.

    Directory of Open Access Journals (Sweden)

    Shuxian Jiang

    2010-03-01

    Full Text Available Mammary stem cells are maintained within specific microenvironments and recruited throughout lifetime to reconstitute de novo the mammary gland. Mammary stem cells have been isolated through the identification of specific cell surface markers and in vivo transplantation into cleared mammary fat pads. Accumulating evidence showed that during the reformation of mammary stem cell niches by dispersed epithelial cells in the context of the intact epithelium-free mammary stroma, non-mammary epithelial cells may be sequestered and reprogrammed to perform mammary epithelial cell functions and to adopt mammary epithelial characteristics during reconstruction of mammary epithelium in regenerating mammary tissue in vivo.To examine whether other types of progenitor cells are able to contribute to mammary branching morphogenesis, we examined the potential of murine embryonic stem (mES cells, undergoing hematopoietic differentiation, to support mammary reconstitution in vivo. We observed that cells from day 14 embryoid bodies (EBs under hematopoietic differentiation condition, but not supernatants derived from these cells, when transplanted into denuded mammary fat pads, were able to contribute to both the luminal and myoepithelial lineages in branching ductal structures resembling the ductal-alveolar architecture of the mammary tree. No teratomas were observed when these cells were transplanted in vivo.Our data provide evidence for the dominance of the tissue-specific mammary stem cell niche and its role in directing mES cells, undergoing hematopoietic differentiation, to reprogram into mammary epithelial cells and to promote mammary epithelial morphogenesis. These studies should also provide insights into regeneration of damaged mammary gland and the role of the mammary microenvironment in reprogramming cell fate.

  13. Microencapsulation technology: a powerful tool for human embryonic stem cells expansion and cryopreservation

    OpenAIRE

    Correia, Cláudia Susana Pedreira

    2010-01-01

    Dissertation presented to obtain a Master degree in Biotechnology at the Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia Human embryonic stem cells (hESCs) are known by their ability to either self-renewal and differentiate into any adult cell type. These properties confer to hESCs a huge applicability for cell therapy, tissue engineering and drug screening. However, successful implementation of hESCs-based technologies requires the production of large numbers of well chara...

  14. Derivation of human embryonic stem cell from spinal muscular atrophy patient

    Directory of Open Access Journals (Sweden)

    Pingyuan Xie

    2016-03-01

    Full Text Available We established a human embryonic stem cell (hESC line chHES-427 from the abnormal embryo carrying homozygous deletion of exon 7 of survival motor neuron gene (SMN. This cell line maintained a normal karyotype 46, XX during long-term culture. Further characteristic analysis suggested that the cells expressed the pluripotency-related markers and had the capacity to differentiate into the derivatives from the three germ layers in vitro.

  15. A single-cell and feeder-free culture system for monkey embryonic stem cells.

    Science.gov (United States)

    Ono, Takashi; Suzuki, Yutaka; Kato, Yosuke; Fujita, Risako; Araki, Toshihiro; Yamashita, Tomoko; Kato, Hidemasa; Torii, Ryuzo; Sato, Naoya

    2014-01-01

    Primate pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold great potential for research and application in regenerative medicine and drug discovery. To maximize primate PSC potential, a practical system is required for generating desired functional cells and reproducible differentiation techniques. Much progress regarding their culture systems has been reported to date; however, better methods would still be required for their practical use, particularly in industrial and clinical fields. Here we report a new single-cell and feeder-free culture system for primate PSCs, the key feature of which is an originally formulated serum-free medium containing FGF and activin. In this culture system, cynomolgus monkey ESCs can be passaged many times by single-cell dissociation with traditional trypsin treatment and can be propagated with a high proliferation rate as a monolayer without any feeder cells; further, typical PSC properties and genomic stability can be retained. In addition, it has been demonstrated that monkey ESCs maintained in the culture system can be used for various experiments such as in vitro differentiation and gene manipulation. Thus, compared with the conventional culture system, monkey ESCs grown in the aforementioned culture system can serve as a cell source with the following practical advantages: simple, stable, and easy cell maintenance; gene manipulation; cryopreservation; and desired differentiation. We propose that this culture system can serve as a reliable platform to prepare primate PSCs useful for future research and application.

  16. Production of stable GFP-expressing neural cells from P19 embryonal carcinoma stem cells.

    Science.gov (United States)

    Shirzad, Hedayatollah; Esmaeili, Fariba; Bakhshalizadeh, Shabnam; Ebrahimie, Marzieh; Ebrahimie, Esmaeil

    2017-04-01

    Murine P19 embryonal carcinoma (EC) cells are convenient to differentiate into all germ layer derivatives. One of the advantages of P19 cells is that the exogenous DNA can be easily inserted into them. Here, at the first part of this study, we generated stable GFP-expressing P19 cells (P19-GFP+). FACS and western-blot analysis confirmed stable expression of GFP in the cells. We previously demonstrated the efficient induction of neuronal differentiation from mouse ES and EC cells by application of a neuroprotective drug, selegiline In the second part of this study selegiline was used to induce differentiation of P19-GFP+ into stable GFP-expressing neuron-like cells. Cresyl violet staining confirmed neuronal morphology of the differentiated cells. Furthermore, real-time PCR and immunoflourescence approved the expression of neuron specific markers. P19-GFP+ cells were able to survive, migrate and integrated into host tissues when transplanted to developing chick embryo CNS. The obtained live GFP-expressing cells can be used as an abundant source of developmentally pluripotent material for transplantation studies, investigating the cellular and molecular aspects of early differentiation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Fingerprinting of neurotoxic compounds using a mouse embryonic stem cell dual luminescence reporter assay

    NARCIS (Netherlands)

    Colaianna, M.; Ilmjärv, S.; Peterson, H.; Ilse Kern, I.; Julien, S.; Baquié, M.; allocca, G.; Bosgra, S.; Sachinidis, A.; Hengstler, J.G.; Leist, M.; Krause, K.H.

    2017-01-01

    Identification of neurotoxic drugs and environmental chemicals is an important challenge. However, only few tools to address this topic are available. The aim of this study was to develop a neurotoxicity/developmental neurotoxicity (DNT) test system, using the pluripotent mouse embryonic stem cell

  18. Redox Disrupting Potential of ToxCast™Chemicals Ranked by Activity in Mouse Embryonic Stem Cells

    Science.gov (United States)

    Little is known regarding the adverse outcome pathways responsible for developmental toxicity following exposure to chemicals. An evaluation of Toxoast™ Phase I chemicals in an adherent mouse embryonic stem cell (mESC) assay revealed a redox sensitive pathway that correlated with...

  19. Generation of a homozygous GBA deletion human embryonic stem cell line

    Directory of Open Access Journals (Sweden)

    Anna Lisa Gündner

    2017-08-01

    Full Text Available We describe the generation of a biallelic GBA deletion human embryonic stem cell line using zinc finger nuclease-mediated gene targeting. The homozygous targeting of exon 4 of the GBA locus leads to a complete loss of glucocerebrosidase (GCase protein expression.

  20. Cigarette smoking during early pregnancy reduces the number of embryonic germ and somatic cells

    DEFF Research Database (Denmark)

    Mamsen, Linn; Lutterodt, M C; Andersen, Elisabeth Anne Wreford

    2010-01-01

    BACKGROUND: Cigarette smoking during pregnancy is associated with negative reproductive consequences for male fetuses in adult life such as reduced testicular volume and sperm concentration. The present study evaluates the number of germ and somatic cells present in human embryonic first-trimeste......BACKGROUND: Cigarette smoking during pregnancy is associated with negative reproductive consequences for male fetuses in adult life such as reduced testicular volume and sperm concentration. The present study evaluates the number of germ and somatic cells present in human embryonic first...... = 0.004] and somatic cells by 37% (95% CI 59-3%, P = 0.023) was observed in testes prenatally exposed to maternal cigarette smoking, compared with unexposed. The effect of maternal smoking was dose-dependent being higher in the heavy smokers and remained consistent after adjusting for possible...... confounders such as alcohol and coffee consumption (P = 0.002). The number of germ cells in embryonic gonads, irrespective of gender, was also significantly reduced by 41% (95% CI 58-19%, P = 0.001) in exposed versus non-exposed embryonic gonads. CONCLUSIONS: Prenatal exposure to maternal cigarette smoke...

  1. Partial promoter substitutions generating transcriptional sentinels of diverse signaling pathways in embryonic stem cells and mice

    DEFF Research Database (Denmark)

    Serup, Palle; Gustavsen, Carsten; Klein, Tino

    2012-01-01

    extracellular signals. We thereby created an allelic series of embryonic stem cells and mice, each containing a signal-responsive sentinel with different fluorescent reporters that respond with sensitivity and specificity to retinoic acids, bone morphogenic proteins, activin A, Wnts or Notch, and that can...

  2. REDOX DISRUPTING POTENTIAL OF TOXCAST CHEMICALS RANKED BY ACTIVITY IN MOUSE EMBRYONIC STEM CELLS

    Science.gov (United States)

    To gain insight regarding the adverse outcome pathways leading to developmental toxicity following exposure to chemicals, we evaluated ToxCast™ Phase I chemicals in an adherent mouse embryonic stem cell (mESC) assay and identified a redox sensitive pathway that correlated with al...

  3. Photo-transfection and selective optical differentiation of embryonic stem cells

    CSIR Research Space (South Africa)

    Mthunzi, P

    2010-09-01

    Full Text Available and differentiation programs are operated and regulated at the genetic level. Genetic manipulation such as delivery of exogenous gene expression or knockout with small interfering RNA (siRNA) is relatively rare in mouse embryonic (ES) cells. However, genetic species...

  4. Erk signaling suppresses embryonic stem cell self-renewal to specify endoderm

    DEFF Research Database (Denmark)

    Hamilton, William B; Brickman, Joshua M

    2014-01-01

    Fgf signaling via Erk activation has been associated with both neural induction and the generation of a primed state for the differentiation of embryonic stem cells (ESCs) to all somatic lineages. To dissect the role of Erk in both ESC self-renewal and lineage specification, we explored...

  5. Functionally deficient neuronal differentiation of mouse embryonic neural stem cells in vitro

    NARCIS (Netherlands)

    Balasubramaniyan, [No Value; de Haas, AH; Bakels, R; Koper, A; Boddeke, HWGM; Copray, JM

    Embryonic mouse neural stem cells (NSCs) were isolated from E14 mice, multiplied in medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) and plated in laminin-coated wells in basic serum-free neurobasal medium. After 7 days in vitro, approximately 20% of the

  6. Utx Is Required for Proper Induction of Ectoderm and Mesoderm during Differentiation of Embryonic Stem Cells

    DEFF Research Database (Denmark)

    Morales Torres, Cristina; Laugesen, Anne; Helin, Kristian

    2013-01-01

    for the activation of lineage choice genes in response to developmental signals. To further understand the function of Utx in pluripotency and differentiation we generated Utx knockout embryonic stem cells (ESCs). Here we show that Utx is not required for the proliferation of ESCs, however, Utx contributes......Embryonic development requires chromatin remodeling for dynamic regulation of gene expression patterns to ensure silencing of pluripotent transcription factors and activation of developmental regulators. Demethylation of H3K27me3 by the histone demethylases Utx and Jmjd3 is important...

  7. Transcriptomic changes in mouse embryonic stem cells exposed to thalidomide during spontaneous differentiation

    Directory of Open Access Journals (Sweden)

    Xiugong Gao

    2015-09-01

    Full Text Available Thalidomide is a potent developmental toxicant that induces a range of birth defects, notably severe limb malformations. To unravel the molecular mechanisms underpinning the teratogenic effects of thalidomide, we used microarrays to study transcriptomic changes induced by thalidomide in an in vitro model based on the differentiation of mouse embryonic stem cells (mESCs, and published the major findings in a research article entitled “Thalidomide induced early gene expression perturbations indicative of human embryopathy in mouse embryonic stem cells” [1]. The data presented herein contains complementary information related to the aforementioned research article.

  8. Molecular Biomarkers for Embryonic and Adult Neural Stem Cell and Neurogenesis

    Science.gov (United States)

    2015-01-01

    The procedure of neurogenesis has made numerous achievements in the past decades, during which various molecular biomarkers have been emerging and have been broadly utilized for the investigation of embryonic and adult neural stem cell (NSC). Nevertheless, there is not a consistent and systematic illustration to depict the functional characteristics of the specific markers expressed in distinct cell types during the different stages of neurogenesis. Here we gathered and generalized a series of NSC biomarkers emerging during the procedures of embryonic and adult neural stem cell, which may be used to identify the subpopulation cells with distinguishing characters in different timeframes of neurogenesis. The identifications of cell patterns will provide applications to the detailed investigations of diverse developmental cell stages and the extents of cell differentiation, which will facilitate the tracing of cell time-course and fate determination of specific cell types and promote the further and literal discoveries of embryonic and adult neurogenesis. Meanwhile, via the utilization of comprehensive applications under the aiding of the systematic knowledge framework, researchers may broaden their insights into the derivation and establishment of novel technologies to analyze the more detailed process of embryogenesis and adult neurogenesis. PMID:26421301

  9. Quality Assurance in Stem Cell Banking: Emphasis on Embryonic and Induced Pluripotent Stem Cell Banking.

    Science.gov (United States)

    Kallur, Therése; Blomberg, Pontus; Stenfelt, Sonya; Tryggvason, Kristian; Hovatta, Outi

    2017-01-01

    For quality assurance (QA) in stem cell banking, a planned system is needed to ensure that the banked products, stem cells, meet the standards required for research, clinical use, and commercial biotechnological applications. QA is process oriented, avoids, or minimizes unacceptable product defects, and particularly encompasses the management and operational systems of the bank, as well as the ethical and legal frameworks. Quality control (QC ) is product oriented and therefore ensures the stem cells of a bank are what they are expected to be. Testing is for controlling, not assuring, product quality, and is therefore a part of QC , not QA. Like QA, QC is essential for banking cells for quality research and translational application (Schwartz et al., Lancet 379:713-720, 2012). Human embryonic stem cells (hESCs), as cells derived from donated supernumerary embryos from in vitro fertilization (IVF) therapy, are different from other stem cell types in resulting from an embryo that has had two donors . This imposes important ethical and legal constraints on the utility of the cells, which, together with quite specific culture conditions, require special attention in the QA system. Importantly, although the origin and derivation of induced pluripotent stem cells (iPSCs ) differ from that of hESCs, many of the principles of QA for hESC banking are applicable to iPSC banking (Stacey et al., Cell Stem Cell 13:385-388, 2013). Furthermore, despite differences between the legal and regulatory frameworks for hESC and iPSC banking between different countries, the requirements for QA are being harmonized (Stacey et al., Cell Stem Cell 13:385-388, 2013; International Stem Cell Banking Initiative, Stem Cell Rev 5:301-314, 2009).

  10. Distribution of microglial cells in the cerebral hemispheres of embryonic and neonatal chicks

    Directory of Open Access Journals (Sweden)

    Ignácio A.R.

    2005-01-01

    Full Text Available The distribution, morphology and morphometry of microglial cells in the chick cerebral hemispheres from embryonic day 4 (E4 to the first neonatal day (P1 were studied by histochemical labeling with a tomato (Lycopersicon esculentum lectin. The histochemical analysis revealed lectin-reactive cells in the nervous parenchyma on day E4. Between E4 (5.7 ± 1.35 mm length and E17 (8.25 ± 1.2 mm length, the lectin-reactive cells were identified as ameboid microglia and observed starting from the subventricular layer, distributed throughout the mantle layer and in the proximity of the blood vessels. After day E13, the lectin-reactive cells exhibited elongated forms with small branched processes, and were considered primitive ramified microglia. Later, between E18 (5.85 ± 1.5 mm cell body length and P1 (3.25 ± 0.6 mm cell body length, cells with more elongated branched processes were observed, constituting the ramified microglia. Our findings provide additional information on the migration and differentiation of microglial cells, whose ramified form is observed at the end of embryonic development. The present paper focused on the arrangement of microglial cells in developing cerebral hemispheres of embryonic and neonatal chicks, which are little studied in the literature. Details of morphology, morphometry and spatial distribution of microglial cells contributed to the understanding of bird and mammal central nervous system ontogeny. Furthermore, the identification and localization of microglial cells during the normal development could be used as a morphological guide for embryonic brain injury researches.

  11. The ability of mouse nuclear transfer embryonic stem cells to differentiate into primordial germ cells

    Directory of Open Access Journals (Sweden)

    Vahid Mansouri

    2015-06-01

    Full Text Available Nuclear transfer embryonic stem cells (ntESCs show stem cell characteristics such as pluripotency but cause no immunological disorders. Although ntESCs are able to differentiate into somatic cells, the ability of ntESCs to differentiate into primordial germ cells (PGCs has not been examined. In this work, we examined the capacity of mouse ntESCs to differentiate into PGCs in vitro. ntESCs aggregated to form embryoid bodies (EB in EB culture medium supplemented with bone morphogenetic protein 4(BMP4 as the differentiation factor. The expression level of specific PGC genes was compared at days 4 and 8 using real time PCR. Flow cytometry and immunocytochemical staining were used to detect Mvh as a specific PGC marker. ntESCs expressed particular genes related to different stages of PGC development. Flow cytometry and immunocytochemical staining confirmed the presence of Mvh protein in a small number of cells. There were significant differences between cells that differentiated into PGCs in the group treated with Bmp4 compared to non-treated cells. These findings indicate that ntESCs can differentiate into putative PGCs. Improvement of ntESC differentiation into PGCs may be a reliable means of producing mature germ cells.

  12. Human amniotic epithelial cells as feeder layer to derive and maintain human embryonic stem cells from poor-quality embryos

    Directory of Open Access Journals (Sweden)

    Daniela Ávila-González

    2015-09-01

    Full Text Available Data from the literature suggest that human embryonic stem cell (hESC lines used in research do not genetically represent all human populations. The derivation of hESC through conventional methods involve the destruction of viable human embryos, as well the use of mouse embryonic fibroblasts as a feeder layer, which has several drawbacks. We obtained the hESC line (Amicqui-1 from poor-quality (PQ embryos derived and maintained on human amniotic epithelial cells (hAEC. This line displays a battery of markers of pluripotency and we demonstrated the capacity of these cells to produce derivates of the three germ layers.

  13. The importance of HLA-G expression in embryos, trophoblast cells, and embryonic stem cells.

    Science.gov (United States)

    Rizzo, Roberta; Vercammen, Martine; van de Velde, Hilde; Horn, Peter A; Rebmann, Vera

    2011-02-01

    The nonclassical HLA-G molecule is a trophoblast-specific molecule present in almost every pregnancy. It differs from classical HLA class I molecules by the low degree of allelic variants and the high diversity of protein structures. HLA-G is reported to be a tolerogenic molecule that acts on cells of both innate and adaptive immunity. At the maternal-fetal interface HLA-G seems to be responsible largely for the reprogramming of local maternal immune response. This review will focus on the HLA-G gene expression profile in pregnancy, in preimplantation embryos, and in human embryonic stem cells with emphasis on the structural diversity of the HLA-G protein and its potential functional and diagnostic implications.

  14. Laser-induced fusion of human embryonic stem cells with optical tweezers

    Energy Technology Data Exchange (ETDEWEB)

    Chen Shuxun; Wang Xiaolin; Sun Dong [Department of Mechanical and Biomedical Engineering, City University of Hong Kong (Hong Kong); Cheng Jinping; Han Cheng, Shuk [Department of Biology and Chemistry, City University of Hong Kong (Hong Kong); Kong, Chi-Wing [Stem Cell and Regenerative Medicine Consortium, and Departments of Medicine and Physiology, LKS Faculty of Medicine, University of Hong Kong (Hong Kong); Li, Ronald A. [Stem Cell and Regenerative Medicine Consortium, and Departments of Medicine and Physiology, LKS Faculty of Medicine, University of Hong Kong (Hong Kong); Center of Cardiovascular Research, Mount Sinai School of Medicine, New York, New York 10029 (United States)

    2013-07-15

    We report a study on the laser-induced fusion of human embryonic stem cells (hESCs) at the single-cell level. Cells were manipulated by optical tweezers and fused under irradiation with pulsed UV laser at 355 nm. Successful fusion was indicated by green fluorescence protein transfer. The influence of laser pulse energy on the fusion efficiency was investigated. The fused products were viable as gauged by live cell staining. Successful fusion of hESCs with somatic cells was also demonstrated. The reported fusion outcome may facilitate studies of cell differentiation, maturation, and reprogramming.

  15. Stage-specific embryonic antigen: determining expression in canine glioblastoma, melanoma, and mammary cancer cells.

    Science.gov (United States)

    Lin, Weiming; Modiano, Jaime F; Ito, Daisuke

    2017-03-30

    The expression of stage-specific embryonic antigens (SSEAs) was determined in several types of canine cancer cells. Flow cytometry showed SSEA-1 expression in glioblastoma, melanoma, and mammary cancer cells, although none expressed SSEA-3 or SSEA-4. Expression of SSEA-1 was not detected in lymphoma, osteosarcoma, or hemangiosarcoma cell lines. Relatively stable SSEA-1 expression was observed between 24 and 72 h of culture. After 8 days in culture, sorted SSEA-1(-) and SSEA-1(+) cells re-established SSEA-1 expression to levels comparable to those observed in unsorted cells. Our results document, for the first time, the expression of SSEA-1 in several canine cancer cell lines.

  16. Suppression of Th1-mediated autoimmunity by embryonic stem cell-derived dendritic cells.

    Directory of Open Access Journals (Sweden)

    Tokunori Ikeda

    Full Text Available We herein demonstrate the immune-regulatory effect of embryonic stem cell-derived dendritic cells (ES-DCs using two models of autoimmune disease, namely non-obese diabetic (NOD mice and experimental autoimmune encephalomyelitis (EAE. Treatment of pre-diabetic NOD mice with ES-DCs exerted almost complete suppression of diabetes development during the observation period for more than 40 weeks. The prevention of diabetes by ES-DCs was accompanied with significant reduction of insulitis and decreased number of Th1 and Th17 cells in the spleen. Development of EAE was also inhibited by the treatment with ES-DCs, and the therapeutic effect was obtained even if ES-DCs were administrated after the onset of clinical symptoms. Treatment of EAE-induced mice with ES-DCs reduced the infiltration of inflammatory cells into the spinal cord and suppressed the T cell response to the myelin antigen. Importantly, the ES-DC treatment did not affect T cell response to an exogenous antigen. As the mechanisms underlying the reduction of the number of infiltrating Th1 cells, we observed the inhibition of differentiation and proliferation of Th1 cells by ES-DCs. Furthermore, the expression of VLA-4α on Th1 cells was significantly inhibited by ES-DCs. Considering the recent advances in human induced pluripotent stem cell-related technologies, these results suggest a clinical application for pluripotent stem cell-derived dendritic cells as a therapy for T cell-mediated autoimmune diseases.

  17. Directed Migration of Embryonic Stem Cell-derived Neural Cells In An Applied Electric Field

    Science.gov (United States)

    Weiss, Mark; Yao, Li

    2014-01-01

    Spinal cord injury or diseases, such as amyotrophic lateral sclerosis, can cause the loss of motor neurons and therefore results in the paralysis of muscles. Stem cells may improve functional recovery by promoting endogenous regeneration, or by directly replacing neurons. Effective directional migration of grafted neural cells to reconstruct functional connections is crucial in the process. Steady direct current electric fields (EFs) play an important role in the development of the central nervous system. A strong biological effect of EFs is the induction of directional cell migration. In this study, we investigated the guided migration of embryonic stem cell (ESC) derived presumptive motor neurons in an applied EF. The dissociated mouse ESC derived presumptive motor neurons or embryoid bodies were subjected to EFs stimulation and the cell migration was studied. We found that the migration of neural precursors from embryoid bodies was toward cathode pole of applied EFs. Single motor neurons migrated to the cathode of the EFs and reversal of EFs poles reversed their migration direction. The directedness and displacement of cathodal migration became more significant when the field strength was increased from 50 mV/mm to 100 mV/mm. EFs stimulation did not influence the cell migration velocity. Our work suggests that EFs may serve as a guidance cue to direct grafted cell migration in vivo. PMID:24804615

  18. Gene-regulatory interactions in embryonic stem cells represent cell-type specific gene regulatory programs.

    Science.gov (United States)

    Ha, Misook; Hong, Soondo

    2017-10-13

    Pluripotency, the ability of embryonic stem cells to differentiate into specialized cell types, is determined by ESC-specific gene regulators such as transcription factors and chromatin modification factors. It is not well understood how ESCs are poised for differentiation, however, and methods are needed for prognosis of the molecular changes in the differentiation of ESCs into specific organs. We describe a new approach to infer cell-type specific gene regulatory programs based on gene regulatory interactions in ESCs. Our method infers the molecular logic of gene regulatory mechanisms by mapping the position-specific combinatory patterns of numerous regulators in ESCs into cell-type specific gene regulations. We validate the proposed approach by recapitulating the RNA-seq and microarray data of neuronal progenitor cells, adult liver cells, and ESCs from the integrated patterns of diverse gene regulators in ESCs. We find that the collective functions of diverse gene regulators in ESCs represent distinct gene regulatory programs in specialized cell types. Our new approach expands our understanding of the differential gene regulatory information in developments encoded in regulatory networks of ESCs. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. Generation of retinal pigment epithelial cells from human embryonic stem cell-derived spherical neural masses.

    Science.gov (United States)

    Cho, Myung Soo; Kim, Sang Jin; Ku, Seung-Yup; Park, Jung Hyun; Lee, Haksup; Yoo, Dae Hoon; Park, Un Chul; Song, Seul Ae; Choi, Young Min; Yu, Hyeong Gon

    2012-09-01

    Dysfunction and loss of retinal pigment epithelium (RPE) are major pathologic changes observed in various retinal degenerative diseases such as aged-related macular degeneration. RPE generated from human pluripotent stem cells can be a good candidate for RPE replacement therapy. Here, we show the differentiation of human embryonic stem cells (hESCs) toward RPE with the generation of spherical neural masses (SNMs), which are pure masses of hESCs-derived neural precursors. During the early passaging of SNMs, cystic structures arising from opened neural tube-like structures showed pigmented epithelial morphology. These pigmented cells were differentiated into functional RPE by neuroectodermal induction and mechanical purification. Most of the differentiated cells showed typical RPE morphologies, such as a polygonal-shaped epithelial monolayer, and transmission electron microscopy revealed apical microvilli, pigment granules, and tight junctions. These cells also expressed molecular markers of RPE, including Mitf, ZO-1, RPE65, CRALBP, and bestrophin. The generated RPE also showed phagocytosis of isolated bovine photoreceptor outer segment and secreting pigment epithelium-derived factor and vascular endothelial growth factor. Functional RPE could be generated from SNM in our method. Because SNMs have several advantages, including the capability of expansion for long periods without loss of differentiation capability, easy storage and thawing, and no need for feeder cells, our method for RPE differentiation may be used as an efficient strategy for generating functional RPE cells for retinal regeneration therapy. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. High-Efficient Transfection of Human Embryonic Stem Cells by Single-Cell Plating and Starvation.

    Science.gov (United States)

    Liu, Hui; Ren, Caiping; Zhu, Bin; Wang, Lei; Liu, Weidong; Shi, Jia; Lin, Jianxing; Xia, Xiaomeng; Zeng, Fei; Chen, Jiawen; Jiang, Xingjun

    2016-03-15

    Nowadays, the low efficiency of small interfering RNA (siRNA) or plasmid DNA (pDNA) transfection is a critical issue in genetic manipulation of human embryonic stem (hES) cells. Development of an efficient transfection method for delivery of siRNAs and plasmids into hES cells becomes more and more imperative. In this study, we tried to modify the traditional transfection protocol by introducing two crucial processes, single-cell plating and starvation, to increase the transfection efficiency in hES cells. Furthermore, we comparatively examined the transfection efficiency of some commercially available siRNA or pDNA transfection reagents in hES cells. Our results showed that the new developed method markedly enhanced the transfection efficiency without influencing the proliferation and pluripotency of hES cells. Lipofectamine RNAiMAX exhibited much higher siRNA transfection efficiency than the other reagents, and FuGENE HD was identified as the best suitable reagent for efficient pDNA transfection of hES cells among the tested reagents.

  1. Directed migration of embryonic stem cell-derived neural cells in an applied electric field.

    Science.gov (United States)

    Li, Yongchao; Weiss, Mark; Yao, Li

    2014-10-01

    Spinal cord injury or diseases, such as amyotrophic lateral sclerosis, can cause the loss of motor neurons and therefore results in the paralysis of muscles. Stem cells may improve functional recovery by promoting endogenous regeneration, or by directly replacing neurons. Effective directional migration of grafted neural cells to reconstruct functional connections is crucial in the process. Steady direct current electric fields (EFs) play an important role in the development of the central nervous system. A strong biological effect of EFs is the induction of directional cell migration. In this study, we investigated the guided migration of embryonic stem cell (ESC) derived presumptive motor neurons in an applied EF. The dissociated mouse ESC derived presumptive motor neurons or embryoid bodies were subjected to EFs stimulation and the cell migration was studied. We found that the migration of neural precursors from embryoid bodies was toward cathode pole of applied EFs. Single motor neurons migrated to the cathode of the EFs and reversal of EFs poles reversed their migration direction. The directedness and displacement of cathodal migration became more significant when the field strength was increased from 50 mV/mm to 100 mV/mm. EFs stimulation did not influence the cell migration velocity. Our work suggests that EFs may serve as a guidance cue to direct grafted cell migration in vivo.

  2. Nanotopography Promotes Pancreatic Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Kim, Jong Hyun; Kim, Hyung Woo; Cha, Kyoung Je; Han, Jiyou; Jang, Yu Jin; Kim, Dong Sung; Kim, Jong-Hoon

    2016-03-22

    Although previous studies suggest that nanotopographical features influence properties and behaviors of stem cells, only a few studies have attempted to derive clinically useful somatic cells from human pluripotent stem cells using nanopatterned surfaces. In the present study, we report that polystyrene nanopore-patterned surfaces significantly promote the pancreatic differentiation of human embryonic and induced pluripotent stem cells. We compared different diameters of nanopores and showed that 200 nm nanopore-patterned surfaces highly upregulated the expression of PDX1, a critical transcription factor for pancreatic development, leading to an approximately 3-fold increase in the percentage of differentiating PDX1(+) pancreatic progenitors compared with control flat surfaces. Furthermore, in the presence of biochemical factors, 200 nm nanopore-patterned surfaces profoundly enhanced the derivation of pancreatic endocrine cells producing insulin, glucagon, or somatostatin. We also demonstrate that nanopore-patterned surface-induced upregulation of PDX1 is associated with downregulation of TAZ, suggesting the potential role of TAZ in nanopore-patterned surface-mediated mechanotransduction. Our study suggests that appropriate cytokine treatments combined with nanotopographical stimulation could be a powerful tool for deriving a high purity of desired cells from human pluripotent stem cells.

  3. A vector-based system for the differentiation of mouse embryonic stem cells toward germ-line cells

    Directory of Open Access Journals (Sweden)

    Reza Ebrahimzadeh-Vesal

    2014-08-01

    Conclusion: In this study, we demonstrated the in vitro generation of mouse embryonic stem cells to germ cells by using a backbone vector containing the fusion gene Stra8-EGFP. The Stra8 gene is a retinoic acid-responsive protein and is able to regulate meiotic initiation.

  4. Characterizing cancer cells with cancer stem cell-like features in 293T human embryonic kidney cells.

    Science.gov (United States)

    Debeb, Bisrat G; Zhang, Xiaomei; Krishnamurthy, Savitri; Gao, Hui; Cohen, Evan; Li, Li; Rodriguez, Angel A; Landis, Melissa D; Lucci, Anthony; Ueno, Naoto T; Robertson, Fredika; Xu, Wei; Lacerda, Lara; Buchholz, Thomas A; Cristofanilli, Massimo; Reuben, James M; Lewis, Michael T; Woodward, Wendy A

    2010-07-08

    Since the first suggestion of prospectively identifiable cancer stem cells in solid tumors, efforts have been made to characterize reported cancer stem cell surrogates in existing cancer cell lines, and cell lines rich with these surrogates have been used to screen for cancer stem cell targeted agents. Although 293T cells were derived from human embryonic kidney, t