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Sample records for ensheathing cell conditioned

  1. Ensheathing cell-conditioned medium directs the differentiation of human umbilical cord blood cells into aldynoglial phenotype cells.

    PubMed

    Ponce-Regalado, María Dolores; Ortuño-Sahagún, Daniel; Zarate, Carlos Beas; Gudiño-Cabrera, Graciela

    2012-06-01

    Despite their similarities to bone marrow precursor cells (PC), human umbilical cord blood (HUCB) PCs are more immature and, thus, they exhibit greater plasticity. This plasticity is evident by their ability to proliferate and spontaneously differentiate into almost any cell type, depending on their environment. Moreover, HUCB-PCs yield an accessible cell population that can be grown in culture and differentiated into glial, neuronal and other cell phenotypes. HUCB-PCs offer many potential therapeutic benefits, particularly in the area of neural replacement. We sought to induce the differentiation of HUCB-PCs into glial cells, known as aldynoglia. These cells can promote neuronal regeneration after lesion and they can be transplanted into areas affected by several pathologies, which represents an important therapeutic strategy to treat central nervous system damage. To induce differentiation to the aldynoglia phenotype, HUCB-PCs were exposed to different culture media. Mononuclear cells from HUCB were isolated and purified by identification of CD34 and CD133 antigens, and after 12 days in culture, differentiation of CD34+ HUCB-PCs to an aldynoglia phenotypic, but not that of CD133+ cells, was induced in ensheathing cell (EC)-conditioned medium. Thus, we demonstrate that the differentiation of HUCB-PCs into aldynoglia cells in EC-conditioned medium can provide a new source of aldynoglial cells for use in transplants to treat injuries or neurodegenerative diseases.

  2. Immunocytochemical characterisation of olfactory ensheathing cells of zebrafish

    PubMed Central

    Lazzari, Maurizio; Bettini, Simone; Franceschini, Valeria

    2014-01-01

    Continuous lifelong neurogenesis is typical of the vertebrate olfactory system. The regenerative ability of olfactory receptor neurons is dependent on the glial cell type specific to the olfactory pathway, designated ‘olfactory ensheathing cells'. Several studies to date have focused on mammalian olfactory ensheathing cells, owing to their potential roles in cell-based therapy for spinal cord injury repair. However, limited information is available regarding this glial cell type in non-mammalian vertebrates, particularly anamniotes. In the current immunocytochemical study, we analysed the features of olfactory ensheathing cells in the zebrafish, Danio rerio. Fish provide a good model for studying glial cells associated with the olfactory pathway of non-mammalian vertebrates. In particular, zebrafish has numerous valuable features that enable its use as a prime model organism for genetic, neurobiological and developmental studies, as well as toxicology and genomics research. Paraffin sections from decalcified heads of zebrafish were processed immunocytochemically to detect proteins used in the research on mammalian olfactory ensheathing cells, including glial fibrillary acid protein (GFAP), S100, neural cell adhesion molecule (NCAM), polysialylated NCAM (PSA-NCAM), vimentin (VIM), p75NTR and galactin (Gal)-1. Notably, GFAP, S100, NCAM and Gal-1 were clearly observed, whereas no vimentin staining was detected. Weak immunostaining for PSA-NCAM and p75NTR was evident. Moreover the degree of marker expression was not uniform in various tracts of the zebrafish olfactory pathway. The immunostaining patterns of the zebrafish olfactory system are distinct from those of other fish to some extent, suggesting interspecific differences. We also showed that the olfactory pathway of zebrafish expresses markers of mammalian olfactory ensheathing cells. The olfactory systems of vertebrates have similarities but there are also marked variations between them. The issue of whether

  3. Therapeutic Potential of Olfactory Ensheathing Cells and Mesenchymal Stem Cells in Spinal Cord Injuries

    PubMed Central

    Anna, Zadroga; Joanna, Czarzasta; Barczewska, Monika; Wojciech, Maksymowicz

    2017-01-01

    Spinal cord injury (SCI) is a devastating neurological condition that affects individuals worldwide, significantly reducing quality of life, for both patients and their families. In recent years there has been a growing interest in cell therapy potential in the context of spinal cord injuries. The present review aims to discuss and compare the restorative approaches based on the current knowledge, available spinal cord restorative cell therapies, and use of selected cell types. However, treatment options for spinal cord injury are limited, but rehabilitation and experimental technologies have been found to help maintain or improve remaining nerve function in some cases. Mesenchymal stem cells as well as olfactory ensheathing cells seem to show therapeutic impact on damaged spinal cord and might be useful in neuroregeneration. Recent research in animal models and first human trials give patients with spinal cord injuries hope for recovery. PMID:28298927

  4. Therapeutic Potential of Olfactory Ensheathing Cells and Mesenchymal Stem Cells in Spinal Cord Injuries.

    PubMed

    Anna, Zadroga; Katarzyna, Jezierska-Woźniak; Joanna, Czarzasta; Barczewska, Monika; Joanna, Wojtkiewicz; Wojciech, Maksymowicz

    2017-01-01

    Spinal cord injury (SCI) is a devastating neurological condition that affects individuals worldwide, significantly reducing quality of life, for both patients and their families. In recent years there has been a growing interest in cell therapy potential in the context of spinal cord injuries. The present review aims to discuss and compare the restorative approaches based on the current knowledge, available spinal cord restorative cell therapies, and use of selected cell types. However, treatment options for spinal cord injury are limited, but rehabilitation and experimental technologies have been found to help maintain or improve remaining nerve function in some cases. Mesenchymal stem cells as well as olfactory ensheathing cells seem to show therapeutic impact on damaged spinal cord and might be useful in neuroregeneration. Recent research in animal models and first human trials give patients with spinal cord injuries hope for recovery.

  5. The culture of olfactory ensheathing cells (OECs)—a distinct glial cell type

    PubMed Central

    Higginson, Jennifer R.; Barnett, Susan C.

    2011-01-01

    Olfactory ensheathing cells (OECs) have become a popular candidate for the transplant-mediated repair of the damaged CNS. In this review a description is made of the origins of these cells and a historical development of their purification and maintenance in culture. In addition, we illustrate the cellular and molecular characteristics of OECs and emphasise that although they share many properties with Schwann cells, they possess several inherent differences which may allow them to be more beneficial for CNS repair. In summary, OECs are distinct glial cells and the detailed understanding of their biological and molecular properties is essential in ensuring their clinical efficacy after cell transplantation. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair. PMID:20816825

  6. Short Report: Olfactory Ensheathing Cells Promote Differentiation of Neural Stem Cells and Robust Neurite Extension

    PubMed Central

    Sethi, Rosh; Sethi, Roshan; Redmond, Andy

    2014-01-01

    Aims The goal of this study was to gain insight into the signaling between olfactory ensheathing cells (OECs) and neural stem cells (NSCs). We sought to understand the impact of OECs on NSC differentiation and neurite extension and to begin to elucidate the factors involved in these interactions to provide new targets for therapeutic interventions. Materials and Methods We utilized lines of OECs that have been extremely well characterized in vitro and in vivo along with well studied NSCs in gels to determine the impact of the coculture in three dimensions. To further elucidate the signaling, we used conditioned media from the OECs as well as fractioned components on NSCs to determine the molecular weight range of the soluble factors that was most responsible for the NSC behavior. Results We found that the coculture of NSCs and OECs led to robust NSC differentiation and extremely long neural processes not usually seen with NSCs in three dimensional gels in vitro. Through culture of NSCs with fractioned OEC media, we determined that molecules larger than 30 kDa have the greatest impact on the NSC behavior. Conclusions Overall, our findings suggest that cocultures of NSCs and OECs may be a novel combination therapy for neural injuries including spinal cord injury (SCI). Furthermore, we have identified a class of molecules which plays a substantial role in the behavior that provides new targets for investigating pharmacological therapies. PMID:24996386

  7. Selective stimulatory action of olfactory ensheathing glia-conditioned medium on oligodendroglial differentiation, with additional reference to signaling mechanisms.

    PubMed

    Carvalho, Litia A; Vitorino, Louise C; Guimarães, Roberta P M; Allodi, Silvana; de Melo Reis, Ricardo A; Cavalcante, Leny A

    2014-07-04

    We examined the effects of conditioned medium from olfactory ensheathing glia (OEGCM) on the differentiation of oligodendrocytes in mixed cultures of early postnatal hippocampi. Differentiation was judged from the numerical density (ND) of cells immunoreactive to 2'3' cyclic nucleotide 3'phosphodiesterase (CNPase) and O4 antibodies. NDs increased according to inverted-U dose-response curves, particularly for CNPase+ cells (9-fold at optimal dilution) and these changes were blocked by inhibitors of ERK1, p38-MAPK, and PI3K. Our results raise the possibility that OEG secreted factor(s) may counteract demyelination induced by trauma, neurodegenerative diseases, and advanced age, and should stimulate novel methods to deliver these factors and/or potentiating chemicals.

  8. Streptococcus pneumoniae resists intracellular killing by olfactory ensheathing cells but not by microglia

    PubMed Central

    Macedo-Ramos, Hugo; Ruiz-Mendoza, Susana; Mariante, Rafael M.; Guimarães, Erick V.; Quadros-de-Souza, Lucas C.; Paiva, Mauricio M.; Ferreira, Eliane de O.; Pinto, Tatiana C. A.; Teixeira, Lucia M.; Allodi, Silvana; Baetas-da-Cruz, Wagner

    2016-01-01

    Olfactory ensheathing cells (OECs) are a type of specialized glial cell currently considered as having a double function in the nervous system: one regenerative, and another immune. Streptococcus pneumoniae is a major agent of severe infections in humans, including meningitis. It is commonly found in the nasopharynx of asymptomatic carriers, and, under certain still unknown conditions, can invade the brain. We evaluated whether pneumococcal cells recovered from lysed OECs and microglia are able to survive by manipulating the host cell activation. An intracellular-survival assay of S. pneumoniae in OECs showed a significant number of bacterial CFU recovered after 3 h of infection. In contrast, microglia assays resulted in a reduced number of CFU. Electron-microscopy analysis revealed a large number of pneumococci with apparently intact morphology. However, microglia cells showed endocytic vesicles containing only bacterial cell debris. Infection of OEC cultures resulted in continuous NF-κB activation. The IFN-γ-induced increase of iNOS expression was reversed in infected OECs. OECs are susceptible to S. pneumoniae infection, which can suppress their cytotoxic mechanisms in order to survive. We suggest that, in contrast to microglia, OECs might serve as safe targets for pneumococci, providing a more stable environment for evasion of the immune system. PMID:27827453

  9. The protective effect of curcumin in Olfactory Ensheathing Cells exposed to hypoxia.

    PubMed

    Bonfanti, Roberta; Musumeci, Teresa; Russo, Cristina; Pellitteri, Rosalia

    2017-02-05

    Curcumin, a phytochemical component derived from the rhizomes of Curcuma longa, has shown a great variety of pharmacological activities, such as anti-inflammatory, anti-tumor, anti-depression and anti-oxidant activity. Therefore, in the last years it has been used as a therapeutic agent since it confers protection in different neurodegenerative diseases, cerebral ischemia and excitotoxicity. Olfactory Ensheathing Cells (OECs) are glial cells of the olfactory system. They are able to secrete several neurotrophic growth factors, promote axonal growth and support the remyelination of damaged axons. OEC transplantation has emerged as a possible experimental therapy to induce repair of spinal cord injury, even if the functional recovery is still limited. Since hypoxia is a secondary effect in spinal cord injury, this in vitro study investigates the protective effect of curcumin in OECs exposed to hypoxia. Primary OECs were obtained from neonatal rat olfactory bulbs and placed both in normal and hypoxic conditions. Furthermore, some cells were grown with basic Fibroblast Growth Factor (bFGF) and/or curcumin at different concentration and times. The results obtained through immunocytochemical procedures and MTT test show that curcumin stimulates cell viability in OECs grown in normal and hypoxic conditions. Furthermore, the synergistic effect of curcumin and bFGF is the most effective exerting protection on OECs. Since spinal cord injury is often accompanied by secondary insults, such as ischemia or hypoxia, our results suggest that curcumin in combination with bFGF might be considered a possible approach for restoration in injuries.

  10. Endothelin uncouples gap junctions in sustentacular cells and olfactory ensheathing cells of the olfactory mucosa.

    PubMed

    Le Bourhis, Mikaël; Rimbaud, Stéphanie; Grebert, Denise; Congar, Patrice; Meunier, Nicolas

    2014-09-01

    Several factors modulate the first step of odour detection in the rat olfactory mucosa (OM). Among others, vasoactive peptides such as endothelin might play multifaceted roles in the different OM cells. Like their counterparts in the central nervous system, the olfactory sensory neurons are encompassed by different glial-like non-neuronal OM cells; sustentacular cells (SCs) surround their cell bodies, whereas olfactory ensheathing cells (OECs) wrap their axons. Whereas SCs maintain both the structural and ionic integrity of the OM, OECs assure protection, local blood flow control and guiding of olfactory sensory neuron axons toward the olfactory bulb. We previously showed that these non-neuronal OM cells are particularly responsive to endothelin in vitro. Here, we confirmed that the endothelin system is strongly expressed in the OM using in situ hybridization. We then further explored the effects of endothelin on SCs and OECs using electrophysiological recordings and calcium imaging approaches on both in vitro and ex vivo OM preparations. Endothelin induced both robust calcium signals and gap junction uncoupling in both types of cells. This latter effect was mimicked by carbenoxolone, a known gap junction uncoupling agent. However, although endothelin is known for its antiapoptotic effect in the OM, the uncoupling of gap junctions by carbenoxolone was not sufficient to limit the cellular death induced by serum deprivation in OM primary culture. The functional consequence of the endothelin 1-induced reduction of the gap junctional communication between OM non-neuronal cells thus remains to be elucidated.

  11. Single-cell printing to form three-dimensional lines of olfactory ensheathing cells.

    PubMed

    Othon, Christina M; Wu, Xingjia; Anders, Juanita J; Ringeisen, Bradley R

    2008-09-01

    Biological laser printing (BioLP) is a unique tool capable of printing high resolution two- and three-dimensional patterns of living mammalian cells, with greater than 95% viability. These results have been extended to primary cultured olfactory ensheathing cells (OECs), harvested from adult Sprague-Dawley rats. OECs have been found to provide stimulating environments for neurite outgrowth in spinal cord injury models. BioLP is unique in that small load volumes ( approximately microLs) are required to achieve printing, enabling low numbers of OECs to be harvested, concentrated and printed. BioLP was used to form several 8 mm lines of OECs throughout a multilayer hydrogel scaffold. The line width was as low as 20 microm, with most lines comprising aligned single cells. Fluorescent confocal microscopy was used to determine the functionality of the printed OECs, to monitor interactions between printed OECs, and to determine the extent of cell migration throughout the 3D scaffold. High-resolution printing of low cell count, harvested OECs is an important advancement for in vitro study of cell interactions and functionality. In addition, these cell-printed scaffolds may provide an alternative for spinal cord repair studies, as the single-cell patterns formed here are on relevant size scales for neurite outgrowth.

  12. Reelin Regulates the Maturation of Dendritic Spines, Synaptogenesis and Glial Ensheathment of Newborn Granule Cells.

    PubMed

    Bosch, Carles; Masachs, Nuria; Exposito-Alonso, David; Martínez, Albert; Teixeira, Cátia M; Fernaud, Isabel; Pujadas, Lluís; Ulloa, Fausto; Comella, Joan X; DeFelipe, Javier; Merchán-Pérez, Angel; Soriano, Eduardo

    2016-09-13

    The Reelin pathway is essential for both neural migration and for the development and maturation of synaptic connections. However, its role in adult synaptic formation and remodeling is still being investigated. Here, we investigated the impact of the Reelin/Dab1 pathway on the synaptogenesis of newborn granule cells (GCs) in the young-adult mouse hippocampus. We show that neither Reelin overexpression nor the inactivation of its intracellular adapter, Dab1, substantially alters dendritic spine numbers in these neurons. In contrast, 3D-electron microscopy (focused ion beam milling/scanning electron microscope) revealed that dysregulation of the Reelin/Dab1 pathway leads to both transient and permanent changes in the types and morphology of dendritic spines, mainly altering mushroom, filopodial, and branched GC spines. We also found that the Reelin/Dab1 pathway controls synaptic configuration of presynaptic boutons in the dentate gyrus, with its dysregulation leading to a substantial decrease in multi-synaptic bouton innervation. Lastly, we show that the Reelin/Dab1 pathway controls astroglial ensheathment of synapses. Thus, the Reelin pathway is a key regulator of adult-generated GC integration, by controlling dendritic spine types and shapes, their synaptic innervation patterns, and glial ensheathment. These findings may help to better understanding of hippocampal circuit alterations in neurological disorders in which the Reelin pathway is implicated.

  13. Delayed olfactory ensheathing cell transplants reduce nociception after dorsal root injury.

    PubMed

    Wu, Ann; Lauschke, Jenny L; Gorrie, Catherine A; Cameron, Nicholas; Hayward, Ian; Mackay-Sim, Alan; Waite, Phil M E

    2011-05-01

    Injury to cervical dorsal roots mimics the deafferentation component of brachial plexus injury in humans, with intractable neuropathic pain in the deafferented limb being a common consequence. Such lesions are generally not amenable to surgical repair. The use of olfactory ensheathing cells (OECs) for dorsal root repair, via acute transplantation, has been successful in several studies. From a clinical point of view, delayed transplantation of OECs would provide a more realistic timeframe for repair. In this study we investigated the effect of delayed OEC transplantation on functional recovery of skilled forepaw movements and amelioration of neuropathic pain, using a C7 and C8 dorsal root injury rat model previously established in our lab. We found that OEC transplantation to the dorsal horn 1 week after root injury effectively attenuated neuropathic disturbances associated with dorsal root injury, including spontaneous pain behavior, tactile allodynia and thermal hyperalgesia. The sensory controls of complex, goal-oriented skilled reaching and ladder walking, however, were not improved by delayed OEC transplantation. We did not detect any significant influence of transplanted OECs on injury-induced central reorganisation and afferent sprouting. The anti-nociceptive effect mediated by OEC transplants may therefore be explained by alternative mechanisms such as modification of inflammation and astrogliosis. The significant effect of OEC transplants in mitigating neuropathic pain may be clinically useful in intractable pain syndromes arising from deafferentation. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.

  14. Olfactory Ensheathing Cells Rescue Optic Nerve Fibers in a Rat Glaucoma Model

    PubMed Central

    Dai, Chao; Khaw, Peng T.; Yin, Zheng Qin; Li, Daqing; Raisman, Geoffrey; Li, Ying

    2012-01-01

    Purpose: To determine if transplantation of olfactory ensheathing cells (OECs) can reduce loss of optic nerve axons after raised intraocular pressure (IOP) in the rat. Methods: OECs cultured from the adult olfactory mucosa were transplanted into the region of the optic disc. The IOP was raised by injection of magnetic microspheres into the anterior chamber. Results: At 4 weeks after raising the IOP, the transplanted OECs had migrated into the dorsal area of the optic nerve head (ONH) where they surrounded the optic nerve fibers with a non-myelinated ensheathment. The mean amount of damage to the ONH astrocytic area in rats was 51.0% compared with 85.8% in those without OEC transplants (P < 0.02) and the mean loss of axons in the optic nerve was 51.0% compared with 80.3% in the absence of OECs (P < 0.01). Conclusions: OECs transplanted into the region of the ONH of the rat can reduce the loss of axons and the damage to ONH astrocytes caused by raised IOP. Translational Relevance: Confirmation of these preliminary experimental data, further understanding of possible mechanisms of axonal protection by OECs, and the longer-term time course of protection could provide a basis for future human clinical trials of autografted OECs, which would be available from autologous nasal epithelial biopsies. PMID:24049703

  15. Reelin Regulates the Maturation of Dendritic Spines, Synaptogenesis and Glial Ensheathment of Newborn Granule Cells

    PubMed Central

    Bosch, Carles; Masachs, Nuria; Exposito-Alonso, David; Martínez, Albert; Teixeira, Cátia M.; Fernaud, Isabel; Pujadas, Lluís; Ulloa, Fausto; Comella, Joan X.; DeFelipe, Javier; Merchán-Pérez, Angel; Soriano, Eduardo

    2016-01-01

    The Reelin pathway is essential for both neural migration and for the development and maturation of synaptic connections. However, its role in adult synaptic formation and remodeling is still being investigated. Here, we investigated the impact of the Reelin/Dab1 pathway on the synaptogenesis of newborn granule cells (GCs) in the young-adult mouse hippocampus. We show that neither Reelin overexpression nor the inactivation of its intracellular adapter, Dab1, substantially alters dendritic spine numbers in these neurons. In contrast, 3D-electron microscopy (focused ion beam milling/scanning electron microscope) revealed that dysregulation of the Reelin/Dab1 pathway leads to both transient and permanent changes in the types and morphology of dendritic spines, mainly altering mushroom, filopodial, and branched GC spines. We also found that the Reelin/Dab1 pathway controls synaptic configuration of presynaptic boutons in the dentate gyrus, with its dysregulation leading to a substantial decrease in multi-synaptic bouton innervation. Lastly, we show that the Reelin/Dab1 pathway controls astroglial ensheathment of synapses. Thus, the Reelin pathway is a key regulator of adult-generated GC integration, by controlling dendritic spine types and shapes, their synaptic innervation patterns, and glial ensheathment. These findings may help to better understanding of hippocampal circuit alterations in neurological disorders in which the Reelin pathway is implicated. Significance Statement The extracellular protein Reelin has an important role in neurological diseases, including epilepsy, Alzheimer's disease and psychiatric diseases, targeting hippocampal circuits. Here we address the role of Reelin in the development of synaptic contacts in adult-generated granule cells (GCs), a neuronal population that is crucial for learning and memory and implicated in neurological and psychiatric diseases. We found that the Reelin pathway controls the shapes, sizes, and types of dendritic

  16. Olfactory ensheathing cell tumor: A case report and review of the literature

    PubMed Central

    MU, QINGCHUN; GAO, HAIJUN; LIU, PENGFEI; HU, XITONG; ZHENG, XU; LI, PEIWEN; LEI, TING; SUN, YUXUE; ZHAO, GANG; HUANG, HAIYAN; HONG, XINYU

    2015-01-01

    Olfactory ensheathing cell tumors (OECTs) are rare malignancies that occur in the anterior fossa. Little is known with regard to their clinical, radiological and immunohistochemical characteristics. The current study reports the case of a 20-year-old male with an OECT, who presented with a severe headache and a generalized convulsion. Cranial magnetic resonance imaging revealed heterogeneous enhancement of the tumor in the left anterior fossa. Complete removal of the tumor via a left frontal craniotomy was conducted, which relieved the patient's headache; however, this resulted in left-sided anosmia that remained during the two-month follow-up. The tumor was immunopositive for vimentin and S-100, and immunonegative for epithelial membrane antigen and Leu-7. The clinical data, imaging features, intraoperative findings and immunohistochemical characteristics of an additional seven cases reported in the literature are also reviewed and analyzed, providing important clinical information for the pre-operative diagnosis and intraoperative removal of OECTs. PMID:26137016

  17. Globose, cystic olfactory ensheathing cell tumor: A case report and literature review

    PubMed Central

    Liu, Yaoling; Wei, Minghai; Yang, Kang; Tan, Zeshi; Sun, Xu; Li, Xinyu; Che, Ningwei; Luan, Lan; Wang, Guanyu; Wang, Xiaofeng; Sun, Yuqiang; Yin, Jian

    2016-01-01

    Olfactory ensheathing cell tumor (OECT) is one of the most rare intracranial, extra-axial tumors located in the anterior cranial fossa. The present study reports a case of a 34-year-old female patient who presented with a history of hyposmia for 1 year, as well as a gradual dizziness and emotional lability for 2 months. Magnetic resonance imaging of the brain revealed a globose, well-defined cystic mass at the midline of the anterior cranial fossa, which was confirmed as an OECT by histology and was completely resected by bifrontal craniotomy. According to the immunostaining results, the tumor was positive for vimentin and S100 protein, and negative for epithelial membrane antigen, glial fibrillary acidic protein and cluster of differentiation 57 (also known as Leu-7). The presentation, imaging findings, histopathological examination and histogenesis of OECT are discussed in the present study, along with a literature review. PMID:27895759

  18. Influence of Biphasic Stimulation on Olfactory Ensheathing Cells for Neuroprosthetic Devices

    PubMed Central

    Hassarati, Rachelle T.; Foster, L. John R.; Green, Rylie A.

    2016-01-01

    The recent success of olfactory ensheathing cell (OEC) assisted regeneration of injured spinal cord has seen a rising interest in the use of these cells in tissue-engineered systems. Previously shown to support neural cell growth through glial scar tissue, OECs have the potential to assist neural network formation in living electrode systems to produce superior neuroprosthetic electrode surfaces. The following study sought to understand the influence of biphasic electrical stimulation (ES), inherent to bionic devices, on cell survival and function, with respect to conventional metallic and developmental conductive hydrogel (CH) coated electrodes. The CH utilized in this study was a biosynthetic hydrogel consisting of methacrylated poly(vinyl-alcohol) (PVA), heparin and gelatin through which poly(3,4-ethylenedioxythiophene) (PEDOT) was electropolymerised. OECs cultured on Pt and CH surfaces were subjected to biphasic ES. Image-based cytometry yielded little significant difference between the viability and cell cycle of OECs cultured on the stimulated and passive samples. The significantly lower voltages measured across the CH electrodes (147 ± 3 mV) compared to the Pt (317 ± 5 mV), had shown to influence a higher percentage of viable cells on CH (91–93%) compared to Pt (78–81%). To determine the functionality of these cells following electrical stimulation, OECs co-cultured with PC12 cells were found to support neural cell differentiation (an indirect measure of neurotrophic factor production) following ES. PMID:27757072

  19. Generation of three-dimensional multiple spheroid model of olfactory ensheathing cells using floating liquid marbles

    PubMed Central

    Vadivelu, Raja K.; Ooi, Chin H.; Yao, Rebecca-Qing; Tello Velasquez, Johana; Pastrana, Erika; Diaz-Nido, Javier; Lim, Filip; Ekberg, Jenny A. K.; Nguyen, Nam-Trung; St John, James A.

    2015-01-01

    We describe a novel protocol for three-dimensional culturing of olfactory ensheathing cells (OECs), which can be used to understand how OECs interact with other cells in three dimensions. Transplantation of OECs is being trialled for repair of the paralysed spinal cord, with promising but variable results and thus the therapy needs improving. To date, studies of OEC behaviour in a multicellular environment have been hampered by the lack of suitable three-dimensional cell culture models. Here, we exploit the floating liquid marble, a liquid droplet coated with hydrophobic powder and placed on a liquid bath. The presence of the liquid bath increases the humidity and minimises the effect of evaporation. Floating liquid marbles allow the OECs to freely associate and interact to produce OEC spheroids with uniform shapes and sizes. In contrast, a sessile liquid marble on a solid surface suffers from evaporation and the cells aggregate with irregular shapes. We used floating liquid marbles to co-culture OECs with Schwann cells and astrocytes which formed natural structures without the confines of gels or bounding layers. This protocol can be used to determine how OECs and other cell types associate and interact while forming complex cell structures. PMID:26462469

  20. Generation of three-dimensional multiple spheroid model of olfactory ensheathing cells using floating liquid marbles

    NASA Astrophysics Data System (ADS)

    Vadivelu, Raja K.; Ooi, Chin H.; Yao, Rebecca-Qing; Tello Velasquez, Johana; Pastrana, Erika; Diaz-Nido, Javier; Lim, Filip; Ekberg, Jenny A. K.; Nguyen, Nam-Trung; St John, James A.

    2015-10-01

    We describe a novel protocol for three-dimensional culturing of olfactory ensheathing cells (OECs), which can be used to understand how OECs interact with other cells in three dimensions. Transplantation of OECs is being trialled for repair of the paralysed spinal cord, with promising but variable results and thus the therapy needs improving. To date, studies of OEC behaviour in a multicellular environment have been hampered by the lack of suitable three-dimensional cell culture models. Here, we exploit the floating liquid marble, a liquid droplet coated with hydrophobic powder and placed on a liquid bath. The presence of the liquid bath increases the humidity and minimises the effect of evaporation. Floating liquid marbles allow the OECs to freely associate and interact to produce OEC spheroids with uniform shapes and sizes. In contrast, a sessile liquid marble on a solid surface suffers from evaporation and the cells aggregate with irregular shapes. We used floating liquid marbles to co-culture OECs with Schwann cells and astrocytes which formed natural structures without the confines of gels or bounding layers. This protocol can be used to determine how OECs and other cell types associate and interact while forming complex cell structures.

  1. Biofunctionalization of conductive hydrogel coatings to support olfactory ensheathing cells at implantable electrode interfaces.

    PubMed

    Hassarati, Rachelle T; Marcal, Helder; John, L; Foster, R; Green, Rylie A

    2016-05-01

    Mechanical discrepancies between conventional platinum (Pt) electrodes and neural tissue often result in scar tissue encapsulation of implanted neural recording and stimulating devices. Olfactory ensheathing cells (OECs) are a supportive glial cell in the olfactory nervous system which can transition through glial scar tissue while supporting the outgrowth of neural processes. It has been proposed that this function can be used to reconnect implanted electrodes with the target neural pathways. Conductive hydrogel (CH) electrode coatings have been proposed as a substrate for supporting OEC survival and proliferation at the device interface. To determine an ideal CH to support OECs, this study explored eight CH variants, with differing biochemical composition, in comparison to a conventional Pt electrodes. All CH variants were based on a biosynthetic hydrogel, consisting of poly(vinyl alcohol) and heparin, through which the conductive polymer (CP) poly(3,4-ethylenedioxythiophene) was electropolymerized. The biochemical composition was varied through incorporation of gelatin and sericin, which were expected to provide cell adherence functionality, supporting attachment, and cell spreading. Combinations of these biomolecules varied from 1 to 3 wt %. The physical, electrical, and biological impact of these molecules on electrode performance was assessed. Cyclic voltammetry and electrochemical impedance spectroscopy demonstrated that the addition of these biological molecules had little significant effect on the coating's ability to safely transfer charge. Cell attachment studies, however, determined that the incorporation of 1 wt % gelatin in the hydrogel was sufficient to significantly increase the attachment of OECs compared to the nonfunctionalized CH.

  2. Nose-to-brain delivery: evaluation of polymeric nanoparticles on olfactory ensheathing cells uptake.

    PubMed

    Musumeci, Teresa; Pellitteri, Rosalia; Spatuzza, Michela; Puglisi, Giovanni

    2014-02-01

    The nasal route has received a great deal of attention as a convenient and reliable method for the brain target on administration of drugs. When drugs are loaded into nanoparticles (NPs) the interaction with mucosa transports directly into the brain, skipping the blood-brain barrier and achieving rapid cerebrospinal fluid levels. Poly-lactic acid (PLA), poly-lactic-co-glycolic acid (PLGA), and chitosan (CS) were chosen to prepare NPs. After optimization of CS nanocarriers, our goal was to evaluate the different type of NPs uptake into olfactory ensheathing cells (OECs). We then correlated obtained biological data to zeta potential measurements of cells treated with NPs. Rodhamine-loaded NPs were used to study the uptake of OECs carried out by confocal microscopy at different times (1, 2, and 4 h). Our results showed that uptake of rodhamine-NPs by OECs was time dependent and it was influenced by the carrier charge. Confocal imaging of OECs demonstrated that NPPLGA showed a higher increase in uptake compared with NPPLA and NPCS after 1 h and it increased at 2-4 h. Zeta potential values of treated cells were more amplified with respect to untreated cells. The highest values were showed by unloaded NPPLGA, confirming microscopy data.

  3. Brain-derived Neurotrophic Factor Promotes the Migration of Olfactory Ensheathing Cells Through TRPC Channels.

    PubMed

    Wang, Ying; Teng, Hong-Lin; Gao, Yuan; Zhang, Fan; Ding, Yu-Qiang; Huang, Zhi-Hui

    2016-12-01

    Olfactory ensheathing cells (OECs) are a unique type of glial cells with axonal growth-promoting properties in the olfactory system. Organized migration of OECs is essential for neural regeneration and olfactory development. However, the molecular mechanism of OEC migration remains unclear. In the present study, we examined the effects of brain-derived neurotrophic factor (BDNF) on OEC migration. Initially, the "scratch" migration assay, the inverted coverslip and Boyden chamber migration assays showed that BDNF could promote the migration of primary cultured OECs. Furthermore, BDNF gradient attracted the migration of OECs in single-cell migration assays. Mechanistically, TrkB receptor expressed in OECs mediated BDNF-induced OEC migration, and BDNF triggered calcium signals in OECs. Finally, transient receptor potential cation channels (TRPCs) highly expressed in OECs were responsible for BDNF-induced calcium signals, and required for BDNF-induced OEC migration. Taken together, these results demonstrate that BDNF promotes the migration of cultured OECs and an unexpected finding is that TRPCs are required for BDNF-induced OEC migration. GLIA 2016;64:2154-2165.

  4. Synergetic effects of ciliary neurotrophic factor and olfactory ensheathing cells on optic nerve reparation (complete translation)

    PubMed Central

    Yin, Dan-ping; Chen, Qing-ying; Liu, Lin

    2016-01-01

    At present, there is no effective treatment for the repair of the optic nerve after injury, or improvement of its microenvironment for regeneration. Intravitreally injected ciliary neurotrophic factor (CNTF) and olfactory ensheathing cells (OECs) promote the long-distance regrowth of severed optic nerve fibers after intracranial injury. Here, we examined the efficacy of these techniques alone and in combination, in a rat model of optic nerve injury. We injected condensed OEC suspension at the site of injury, or CNTF into the vitreous body, or both simultaneously. Retrograde tracing techniques showed that 4 weeks postoperatively, the number of surviving retinal ganglion cells and their axonal density in the optic nerve were greater in rats subjected to OEC injection only than in those receiving CNTF injection only. Furthermore, combined OEC + CNTF injection achieved better results than either monotherapy. These findings confirm that OECs are better than CNTF at protecting injured neurons in the eye, but that combined OEC and CNTF therapy is notably more effective than either treatment alone. PMID:27482233

  5. Microencapsulation improves inhibitory effects of transplanted olfactory ensheathing cells on pain after sciatic nerve injury

    PubMed Central

    Zhao, Hao; Yang, Bao-lin; Liu, Zeng-xu; Yu, Qing; Zhang, Wen-jun; Yuan, Keng; Zeng, Hui-hong; Zhu, Gao-chun; Liu, De-ming; Li, Qing

    2015-01-01

    Olfactory bulb tissue transplantation inhibits P2X2/3 receptor-mediated neuropathic pain. However, the olfactory bulb has a complex cellular composition, and the mechanism underlying the action of purified transplanted olfactory ensheathing cells (OECs) remains unclear. In the present study, we microencapsulated OECs in alginic acid, and transplanted free and microencapsulated OECs into the region surrounding the injured sciatic nerve in rat models of chronic constriction injury. We assessed mechanical nociception in the rat models 7 and 14 days after surgery by measuring paw withdrawal threshold, and examined P2X2/3 receptor expression in L4–5 dorsal root ganglia using immunohistochemistry. Rats that received free and microencapsulated OEC transplants showed greater withdrawal thresholds than untreated model rats, and weaker P2X2/3 receptor immunoreactivity in dorsal root ganglia. At 14 days, paw withdrawal threshold was much higher in the microencapsulated OEC-treated animals. Our results confirm that microencapsulated OEC transplantation suppresses P2X2/3 receptor expression in L4–5 dorsal root ganglia in rat models of neuropathic pain and reduces allodynia, and also suggest that transplantation of microencapsulated OECs is more effective than transplantation of free OECs for the treatment of neuropathic pain. PMID:26487865

  6. Electrical regulation of olfactory ensheathing cells using conductive polypyrrole/chitosan polymers.

    PubMed

    Qi, Fengyu; Wang, Yuqing; Ma, Teng; Zhu, Shu; Zeng, Wen; Hu, Xueyu; Liu, Zhongyang; Huang, Jinghui; Luo, Zhuojing

    2013-02-01

    Electrical stimulation (ES) applied to a conductive nerve graft holds the great potential to improve nerve regeneration and functional recovery in the treatment of lengthy nerve defects. A conductive nerve graft can be obtained by a combination of conductive nerve scaffold and olfactory ensheathing cells (OECs), which are known to enhance axonal regeneration and to produce myelin after transplantation. However, when ES is applied through the conductive graft, the impact of ES on OECs has never been investigated. In this study, a biodegradable conductive composite made of conductive polypyrrole (PPy, 2.5%) and biodegradable chitosan (97.5%) was prepared in order to electrically stimulate OECs. The tolerance of OECs to ES was examined by a cell apoptosis assay. The growth of the cells was characterized using DAPI staining and a CCK-8 assay. The mRNA and protein levels of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neural cell adhesion molecule (N-CAM), vascular endothelial growth factor (VEGF) and neurite outgrowth inhibitor-A (NOGO-A) in OECs were assayed by RT-PCR and Western blotting, and the amount of BDNF, NGF, N-CAM, VEGF and NOGO-A secreted was determined by an ELISA assay. The results showed that the PPy/chitosan membranes supported cell adhesion, spreading, and proliferation with or without ES. Interestingly, ES applied through the PPy/chitosan composite dramatically enhanced the expression and secretion of BDNF, NGF, N-CAM and VEGF, but decreased the expression and secretion of NOGO-A when compared with control cells without ES. These findings highlight the possibility of enhancing nerve regeneration in conductive scaffolds through ES increased neurotrophin secretion in OECs.

  7. Low-Dose Curcumin Stimulates Proliferation, Migration and Phagocytic Activity of Olfactory Ensheathing Cells

    PubMed Central

    Tello Velasquez, Johana; Watts, Michelle E.; Todorovic, Michael; Nazareth, Lynnmaria; Pastrana, Erika; Diaz-Nido, Javier; Lim, Filip; Ekberg, Jenny A. K.; Quinn, Ronald J.; John, James A. St

    2014-01-01

    One of the promising strategies for neural repair therapies is the transplantation of olfactory ensheathing cells (OECs) which are the glial cells of the olfactory system. We evaluated the effects of curcumin on the behaviour of mouse OECs to determine if it could be of use to further enhance the therapeutic potential of OECs. Curcumin, a natural polyphenol compound found in the spice turmeric, is known for its anti-cancer properties at doses over 10 µM, and often at 50 µM, and it exerts its effects on cancer cells in part by activation of MAP kinases. In contrast, we found that low-dose curcumin (0.5 µM) applied to OECs strikingly modulated the dynamic morphology, increased the rate of migration by up to 4-fold, and promoted significant proliferation of the OECs. Most dramatically, low-dose curcumin stimulated a 10-fold increase in the phagocytic activity of OECs. All of these potently stimulated behavioural characteristics of OECs are favourable for neural repair therapies. Importantly, low-dose curcumin gave a transient activation of p38 kinases, which is in contrast to the high dose curcumin effects on cancer cells in which these MAP kinases tend to undergo prolonged activation. Low-dose curcumin mediated effects on OECs demonstrate cell-type specific stimulation of p38 and ERK kinases. These results constitute the first evidence that low-dose curcumin can modulate the behaviour of olfactory glia into a phenotype potentially more favourable for neural repair and thereby improve the therapeutic use of OECs for neural repair therapies. PMID:25360677

  8. The mannose receptor is expressed by olfactory ensheathing cells in the rat olfactory bulb.

    PubMed

    Carvalho, Litia A; Nobrega, Alberto F; Soares, Igor D P; Carvalho, Sergio L; Allodi, Silvana; Baetas-da-Cruz, Wagner; Cavalcante, Leny A

    2013-12-01

    Complex carbohydrate structures are essential molecules of infectious bacteria, parasites, and host cells and are involved in cell signaling associated with immune responses, glycoprotein homeostasis, and cell migration. The uptake of mannose-tailed glycans is usually carried out by professional phagocytes to trigger MHC class I- and MHC class II-restricted antigen presentation or, alternatively, to end inflammation. We have detected the mannose receptor (MR) in cultured olfactory ensheathing cells (OECs), so we investigated by flow cytometry whether recently dissociated cells of the olfactory bulb (OB) nerve fiber layer (ONL) could bind a mannosylated ligand (fluorescein conjugate of mannosyl bovine serum albumin; Man/BSA-FITC) in a specific manner. In addition, we estimated the relative proportion of ONL OECs, microglia, and astrocytes, tagged by 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), by the B4 isolectin of Griffonia simplicifonia (IB4), and by glial fibrillary acidic protein (GFAP), respectively, that were Man/BSA-FITC(+) . We also determined by histochemistry and/or immunohistochemistry whether Man/BSA-FITC or an anti-MR antibody (anti-C-terminal MR peptide; anti-cMR) labeled OECs and/or parenchymal microglia. In addition, we confirmed by Western blot with the K1K2 (against the entire MR molecule) antibody that a band of about 180 kDA is expressed in the OB. Our findings are compatible with a prospective sentinel role of OECs against pathogens of the upper airways and/or damage-associated glycidic patterns as well as with homeostasis of OB mannosylated glycoproteins.

  9. Olfactory Ensheathing Cells Express α7 Integrin to Mediate Their Migration on Laminin

    PubMed Central

    Ingram, Norianne T.; Khankan, Rana R.; Phelps, Patricia E.

    2016-01-01

    The unique glia located in the olfactory system, called olfactory ensheathing cells (OECs), are implicated as an attractive choice for transplantation therapy following spinal cord injury because of their pro-regenerative characteristics. Adult OECs are thought to improve functional recovery and regeneration after injury by secreting neurotrophic factors and making cell-to-cell contacts with regenerating processes, but the mechanisms are not well understood. We show first that α7 integrin, a laminin receptor, is highly expressed at the protein level by OECs throughout the olfactory system, i.e., in the olfactory mucosa, olfactory nerve, and olfactory nerve layer of the olfactory bulb. Then we asked if OECs use the α7 integrin receptor directly to promote neurite outgrowth on permissive and neutral substrates, in vitro. We co-cultured α7+/+ and α7lacZ/lacZ postnatal cerebral cortical neurons with α7+/+ or α7lacZ/lacZ OECs and found that genotype did not effect the ability of OECs to enhance neurite outgrowth by direct contact. Loss of α7 integrin did however significantly decrease the motility of adult OECs in transwell experiments. Twice as many α7+/+ OECs migrated through laminin-coated transwells compared to α7+/+ OECs on poly-L-lysine (PLL). This is in contrast to α7lacZ/lacZ OECs, which showed no migratory preference for laminin substrate over PLL. These results demonstrate that OECs express α7 integrin, and that laminin and its α7 integrin receptor contribute to adult OEC migration in vitro and perhaps also in vivo. PMID:27078717

  10. Gene Expression Changes in the Injured Spinal Cord Following Transplantation of Mesenchymal Stem Cells or Olfactory Ensheathing Cells

    PubMed Central

    Torres-Espín, Abel; Hernández, Joaquim; Navarro, Xavier

    2013-01-01

    Transplantation of bone marrow derived mesenchymal stromal cells (MSC) or olfactory ensheathing cells (OEC) have demonstrated beneficial effects after spinal cord injury (SCI), providing tissue protection and improving the functional recovery. However, the changes induced by these cells after their transplantation into the injured spinal cord remain largely unknown. We analyzed the changes in the spinal cord transcriptome after a contusion injury and MSC or OEC transplantation. The cells were injected immediately or 7 days after the injury. The mRNA of the spinal cord injured segment was extracted and analyzed by microarray at 2 and 7 days after cell grafting. The gene profiles were analyzed by clustering and functional enrichment analysis based on the Gene Ontology database. We found that both MSC and OEC transplanted acutely after injury induce an early up-regulation of genes related to tissue protection and regeneration. In contrast, cells transplanted at 7 days after injury down-regulate genes related to tissue regeneration. The most important change after MSC or OEC transplant was a marked increase in expression of genes associated with foreign body response and adaptive immune response. These data suggest a regulatory effect of MSC and OEC transplantation after SCI regarding tissue repair processes, but a fast rejection response to the grafted cells. Our results provide an initial step to determine the mechanisms of action and to optimize cell therapy for SCI. PMID:24146830

  11. Morphological study of a connexin 43-GFP reporter mouse highlights glial heterogeneity, amacrine cells, and olfactory ensheathing cells.

    PubMed

    Theofilas, Panos; Steinhäuser, Christian; Theis, Martin; Derouiche, Amin

    2017-03-30

    Connexin 43 (Cx43) is the main astrocytic connexin and forms the basis of the glial syncytium. The morphology of connexin-expressing cells can be best studied in transgenic mouse lines expressing cytoplasmic fluorescent reporters, since immunolabeling the plaques can obscure the shapes of the individual cells. The Cx43kiECFP mouse generated by Degen et al. (FASEBJ 26:4576, 2012) expresses cytosolic ECFP and has previously been used to establish that Cx43 may not be expressed by all astrocytes within a population, and this can vary in a region-dependent way. To establish this mouse line as a tool for future astrocyte and connexin research, we sought to consolidate reporter authenticity, studying cell types and within-region population heterogeneity. Applying anti-GFP, all cell types related to astroglia were positive-namely, protoplasmic astrocytes in the hippocampus, cortex, thalamus, spinal cord, olfactory bulb, cerebellum with Bergmann glia and astrocytes also in the molecular layer, and retinal Müller cells and astrocytes. Labeled cell types further comprise white matter astrocytes, olfactory ensheathing cells, radial glia-like stem cells, retinal pigment epithelium cells, ependymal cells, and meningeal cells. We furthermore describe a retinal Cx43-expressing amacrine cell morphologically reminiscent of ON-OFF wide-field amacrine cells, representing the first example of a mammalian CNS neuron-expressing Cx43 protein. In double staining with cell type-specific markers (GFAP, S100ß, glutamine synthetase), Cx43 reporter expression in the hippocampus and cortex was restricted to GFAP(+) astrocytes. Altogether, this mouse line is a highly reliable tool for studies of Cx43-expressing CNS cells and astroglial cell morphology. © 2017 Wiley Periodicals, Inc.

  12. Subarachnoid Space Transplantation of Schwann and/or Olfactory Ensheathing Cells Following Severe Spinal Cord Injury Fails to Improve Locomotor Recovery in Rats.

    PubMed

    Nategh, Mohsen; Firouzi, Masoumeh; Naji-Tehrani, Mehdi; Oryadi Zanjani, Leila; Hassannejad, Zahra; Nabian, Mohammad Hosein; Abdollah Zadegan, Shayan; Karimi, Mehrbod; Rahimi-Movaghar, Vafa

    2016-09-01

    Treatment of spinal cord injury by exogenous cells has brought both successful and unsuccessful results. Olfactory ensheathing cells and Schwann cells have been widely used for transplantation purposes. In this study, we investigated the effects of these cells on contused spinal cord by introducing cells into subarachnoid space. Fifty thousand Schwann cells or olfactory ensheathing cells or a mixture of both cell types were transplanted one week after a 3-second clip compression injury at T-9 spinal cord level in rats. Starting from the day one of spinal cord injury, animals were assessed for six months by BBB test and then were sacrificed for immunohistochemistry labeling of the spinal cord injury site. There was no locomotor recovery in any of the treatment groups including controls. Immunohistochemistry assessment indicated positive labeling of P75 and S100 markers in the cell-transplanted groups compared with control. Our data suggest that transplantation of Schwann cells and/or olfactory ensheathing cells into the subarachnoid space does not improve motor recovery in severely injured spinal cord, at least with the number of cells transplanted here. This, however, should not be regarded as an essentially negative outcome, and further studies which consider higher densities of cells are required.

  13. Olfactory Ensheathing Cell Transplantation after a Complete Spinal Cord Transection Mediates Neuroprotective and Immunomodulatory Mechanisms to Facilitate Regeneration

    PubMed Central

    Khankan, Rana R.; Griffis, Khris G.; Haggerty-Skeans, James R.; Zhong, Hui; Roy, Roland R.; Edgerton, V. Reggie

    2016-01-01

    Multiple neural and peripheral cell types rapidly respond to tissue damage after spinal cord injury to form a structurally and chemically inhibitory scar that limits axon regeneration. Astrocytes form an astroglial scar and produce chondroitin sulfate proteoglycans (CSPGs), activate microglia, and recruit blood-derived immune cells to the lesion for debris removal. One beneficial therapy, olfactory ensheathing cell (OEC) transplantation, results in functional improvements and promotes axon regeneration after spinal cord injury. The lack of an OEC-specific marker, however, has limited the investigation of mechanisms underlying their proregenerative effects. We compared the effects of enhanced green fluorescent protein-labeled fibroblast (FB) and OEC transplants acutely after a complete low-thoracic spinal cord transection in adult rats. We assessed the preservation of neurons and serotonergic axons, the levels of inhibitory CSPGs and myelin debris, and the extent of immune cell activation between 1 and 8 weeks postinjury. Our findings indicate that OECs survive longer than FBs post-transplantation, preserve axons and neurons, and reduce inhibitory molecules in the lesion core. Additionally, we show that OECs limit immune-cell activation and infiltration, whereas FBs alter astroglial scar formation and increase immune-cell infiltration and concomitant secondary tissue damage. Administration of cyclosporine-A to enhance graft survival demonstrated that immune suppression can augment OEC contact-mediated protection of axons and neurons during the first 2 weeks postinjury. Collectively, these data suggest that OECs have neuroprotective and immunomodulatory mechanisms that create a supportive environment for neuronal survival and axon regeneration after spinal cord injury. SIGNIFICANCE STATEMENT Spinal cord injury creates physical and chemical barriers to axon regeneration. We used a complete spinal cord transection model and olfactory ensheathing cell (OEC) or

  14. Ultrastructural analysis of olfactory ensheathing cells derived from olfactory bulb and nerve of neonatal and juvenile rats.

    PubMed

    Gómez, Rosa M; Ghotme, Kemel; Botero, Lucía; Bernal, Jaime E; Pérez, Rosalía; Barreto, George E; Bustos, Rosa Helena

    2016-02-01

    Olfactory nerve derived and olfactory bulb derived olfactory ensheathing cells (OECs) have the ability to promote axonal regeneration and remyelination, both of which are essential in a successful cell transplant. Thus, morphological identification of OECs is a key aspect to develop an applicable cell therapy for injuries to the nervous system. However, there is no clear definition regarding which developmental stage or anatomical origin of OECs is more adequate for neural repair. In the present study, an ultrastructural comparison was made between OECs recovered from primary cultures of olfactory nerve and bulb in two developmental stages. The most notorious difference between cells obtained from olfactory nerve and bulb was the presence of indented nuclei in bulb derived OECs, suggesting a greater ability for possible chemotaxis. In neonatal OECs abundant mitochondria, lipid vacuoles, and smooth endoplasmic reticulum were detected, suggesting an active lipid metabolism, probably involved in synthesis of myelin. Our results suggest that neonatal OECs obtained from olfactory bulb have microscopic properties that could make them more suitable for neural repair.

  15. Remyelination of the nonhuman primate spinal cord by transplantation of H-transferase transgenic adult pig olfactory ensheathing cells

    PubMed Central

    Radtke, Christine; Akiyama, Yukinori; Brokaw, Jane; Lankford, Karen L.; Wewetzer, Konstantin; Fodor, William L.; Kocsis, Jeffery D.

    2008-01-01

    Olfactory ensheathing cells (OECs) have been shown to mediate remyelination and to stimulate axonal regeneration in a number of in vivo rodent spinal cord studies. However, whether OECs display similar properties in the primate model has not been tested so far. In the present study, we thus transplanted highly-purified OECs isolated from transgenic pigs expressing the α1,2 fucosyltransferase gene (H-transferase or HT) gene into a demyelinated lesion of the African green monkey spinal cord. Four weeks posttransplantation, robust remyelination was found in 62.5% of the lesion sites, whereas there was virtually no remyelination in the nontransplanted controls. This together with the immunohistochemical demonstration of the grafted cells within the lesioned area confirmed that remyelination was indeed achieved by OECs. Additional in vitro assays demonstrated 1) that the applied cell suspension consisted of >98% OECs, 2) that the majority of the cells expressed the transgene, and 3) that expression of the HT gene reduced complement activation more than twofold compared with the nontransgenic control. This is the first demonstration that xenotransplantation of characterized OECs into the primate spinal cord results in remyelination. PMID:14657003

  16. NTE/PNPLA6 is expressed in mature Schwann cells and is required for glial ensheathment of Remak fibers.

    PubMed

    McFerrin, Janis; Patton, Bruce L; Sunderhaus, Elizabeth R; Kretzschmar, Doris

    2017-02-16

    Neuropathy target esterase (NTE) or patatin-like phospholipase domain containing 6 (PNPLA6) was first linked with a neuropathy occurring after organophosphate poisoning and was later also found to cause complex syndromes when mutated, which can include mental retardation, spastic paraplegia, ataxia, and blindness. NTE/PNPLA6 is widely expressed in neurons but experiments with its Drosophila orthologue Swiss-cheese (SWS) suggested that it may also have glial functions. Investigating whether NTE/PNPLA6 is expressed in glia, we found that NTE/PNPLA6 is expressed by Schwann cells in the sciatic nerve of adult mice with the most prominent expression in nonmyelinating Schwann cells. Within Schwann cells, NTE/PNPLA6 is enriched at the Schmidt-Lanterman incisures and around the nucleus. When analyzing postnatal expression patterns, we did not detect NTE/PNPLA6 in promyelinating Schwann cells, while weak expression was detectable at postnatal day 5 in Schwann cells and increased with their maturation. Interestingly, NTE/PNPLA6 levels were upregulated after nerve crush and localized to ovoids forming along the nerve fibers. Using a GFAP-based knock-out of NTE/PNPLA6, we detected an incomplete ensheathment of Remak fibers whereas myelination did not appear to be affected. These results suggest that NTE/PNPLA6 is involved in the maturation of nonmyelinating Schwann cells during development and de-/remyelination after neuronal injury. Since Schwann cells play an important role in maintaining axonal viability and function, it is therefore likely that changes in Schwann cells contribute to the locomotory deficits and neuropathy observed in patients carrying mutations in NTE.

  17. Global cellular responses to β-methyl-amino-L-alanine (BMAA) by olfactory ensheathing glial cells (OEC).

    PubMed

    Chiu, Alexander S; Braidy, Nady; Marçal, Helder; Welch, Jeffrey H; Gehringer, Michelle M; Guillemin, Gilles J; Neilan, Brett A

    2015-06-01

    This study utilised a proteomics approach to identify any differential protein expression in a glial cell line, rat olfactory ensheathing cells (OECs), treated with the cyanotoxin β-methylamino-l-alanine (BMAA). Five proteins of interest were identified, namely Rho GDP-dissociation inhibitor 1 (RhoGDP1), Nck-associated protein 1 (NCKAP1), voltage-dependent anion-selective channel protein 1 (VDAC1), 3-hydroxyacyl-CoA dehydrogenase type-2 (3hCoAdh2), and ubiquilin-4 (UBQLN4). Four of these candidates, nuclear receptor subfamily 4 group A member 1 (Nur77), cyclophilin A (CyPA), RhoGDP1 and VDAC1, have been reported to be involved in cell growth. A microarray identified UBQLN4, palladin and CyPA, which have been implicated to have roles in excitotoxicity. Moreover, the NCKAP1, UBQLN4, CyPA and 3hCoAdh2 genes have been associated with abnormal protein aggregation. Differential expression of genes involved in mitochondrial activity, Nur77, 3hCoAdh2, VDAC1 and UBQLN4, were also identified. Confirmatory reverse transcription quantitative PCR (RT-qPCR) analysis of transcripts generated from the genes of interest corroborated the differential expression trends identified in the global protein analysis. BMAA induced cell cycle arrest in the G2/M phase of OEC and apoptosis after 48 h at concentrations of 250 μM and 500 μM. Collectively, this work advances our understanding of the mechanism of BMAA-mediated glial-toxicity in vitro.

  18. Fine processes of Nestin-GFP–positive radial glia-like stem cells in the adult dentate gyrus ensheathe local synapses and vasculature

    PubMed Central

    Moss, Jonathan; Gebara, Elias; Sánchez-Pascual, Irene; O’Laoi, Ruadhan; El M’Ghari, Imane; Kocher-Braissant, Jacqueline; Ellisman, Mark H.; Toni, Nicolas

    2016-01-01

    Adult hippocampal neurogenesis relies on the activation of neural stem cells in the dentate gyrus, their division, and differentiation of their progeny into mature granule neurons. The complex morphology of radial glia-like (RGL) stem cells suggests that these cells establish numerous contacts with the cellular components of the neurogenic niche that may play a crucial role in the regulation of RGL stem cell activity. However, the morphology of RGL stem cells remains poorly described. Here, we used light microscopy and electron microscopy to examine Nestin-GFP transgenic mice and provide a detailed ultrastructural reconstruction analysis of Nestin-GFP–positive RGL cells of the dentate gyrus. We show that their primary processes follow a tortuous path from the subgranular zone through the granule cell layer and ensheathe local synapses and vasculature in the inner molecular layer. They share the ensheathing of synapses and vasculature with astrocytic processes and adhere to the adjacent processes of astrocytes. This extensive interaction of processes with their local environment could allow them to be uniquely receptive to signals from local neurons, glia, and vasculature, which may regulate their fate. PMID:27091993

  19. Cell type- and isotype-specific expression and regulation of β-tubulins in primary olfactory ensheathing cells and Schwann cells in vitro.

    PubMed

    Omar, Mohamed; Hansmann, Florian; Kreutzer, Robert; Kreutzer, Mihaela; Brandes, Gudrun; Wewetzer, Konstantin

    2013-05-01

    Olfactory ensheathing cells (OECs) and Schwann cells (SCs) are closely-related cell types with regeneration-promoting properties. Comparative gene expression analysis is particularly relevant since it may explain cell type-specific effects and guide the use of each cell type into special clinical applications. In the present study, we focused on β-tubulin isotype expression in primary adult canine glia as a translational large animal model. β-tubulins so far have been studied mainly in non-neuronal tumors and implied in tumorigenic growth. We show here that primary OECs and SCs expressed βII-V isotype mRNA. Interestingly, βIII-tubulin mRNA and protein expression was high in OECs and low in SCs, while fibroblast growth factor-2 (FGF-2) induced its down-regulation in both cell types to the same extent. This was in contrast to βV-tubulin mRNA which was similarly expressed in both cell types and unaltered by FGF-2. Immunocytochemical analysis revealed that OEC cultures contained a higher percentage of βIII-tubulin-positive cells compared to SC cultures. Addition of FGF-2 reduced the number of βIII-tubulin-positive cells in both cultures and significantly increased the percentage of cells with a multipolar morphology. Taken together, we demonstrate cell type-specific expression (βIII) and isotype-specific regulation (βIII, βV) of β-tubulin isotypes in OECs and SCs. While differential expression of βIII-tubulin in primary glial cell types with identical proliferative behaviour argues for novel functions unrelated to tumorigenic growth, strong βIII-tubulin expression in OECs may help to explain the specific properties of this glial cell type.

  20. Central nervous system lesions that can and those that cannot be repaired with the help of olfactory bulb ensheathing cell transplants.

    PubMed

    Nieto-Sampedro, Manuel

    2003-11-01

    Growth-promoting macroglia (aldynoglia) with growth properties and immunological markers similar to Schwann cells, are found in loci of the mammalian CNS where axon regeneration occurs throughout life, like the olfactory sytem, hypothalamus-hypophysis and the pineal gland. Contrary to Schwann cells, aldynoglia mingle freely with astrocytes and can migrate in brain and spinal cord. Transplantation of cultured and immunopurified olfactory ensheathing cells (OECs) in the spinal cord after multiple central rhizotomy, promoted sensory and central axon growth and partial functional restoration, judging by anatomical, electrophysiological and behavioural criteria. OEC transplants suppressed astrocyte reactivity, thus generally favouring axon growth after a lesion. However, the functional repair promoted by OEC transplants was partial in the best cases, depending on lesion type and location. Cyst formation after photochemical cord lesion was partially prevented but neither the corticospinal tract, interrupted by a mild contusion, nor the sectioned medial longitudinal fascicle, did regrow after OEC transplantation in the injured area.

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

    PubMed Central

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

    2017-01-01

    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. PMID:28146061

  2. Effects of Neural Stem Cell and Olfactory Ensheathing Cell Co-transplants on Tissue Remodelling After Transient Focal Cerebral Ischemia in the Adult Rat.

    PubMed

    Augestad, Ingrid Lovise; Nyman, Axel Karl Gottfrid; Costa, Alex Ignatius; Barnett, Susan Carol; Sandvig, Axel; Håberg, Asta Kristine; Sandvig, Ioanna

    2017-01-24

    Effective transplant-mediated repair of ischemic brain lesions entails extensive tissue remodeling, especially in the ischemic core. Neural stem cells (NSCs) are promising reparative candidates for stroke induced lesions, however, their survival and integration with the host-tissue post-transplantation is poor. In this study, we address this challenge by testing whether co-grafting of NSCs with olfactory ensheathing cells (OECs), a special type of glia with proven neuroprotective, immunomodulatory, and angiogenic effects, can promote graft survival and host tissue remodelling. Transient focal cerebral ischemia was induced in adult rats by a 60-min middle cerebral artery occlusion (MCAo) followed by reperfusion. Ischemic lesions were verified by neurological testing and magnetic resonance imaging. Transplantation into the globus pallidus of NSCs alone or in combination with OECs was performed at two weeks post-MCAo, followed by histological analyses at three weeks post-transplantation. We found evidence of extensive vascular remodelling in the ischemic core as well as evidence of NSC motility away from the graft and into the infarct border in severely lesioned animals co-grafted with OECs. These findings support a possible role of OECs as part of an in situ tissue engineering paradigm for transplant mediated repair of ischemic brain lesions.

  3. Regeneration of nigrostriatal dopaminergic axons after transplantation of olfactory ensheathing cells and fibroblasts prevents fibrotic scar formation at the lesion site.

    PubMed

    Teng, Xichuan; Nagata, Isao; Li, Hong-Peng; Kimura-Kuroda, Junko; Sango, Kazunori; Kawamura, Koki; Raisman, Geoffrey; Kawano, Hitoshi

    2008-11-01

    The fibrotic scar formed after central nervous system injury has been considered an obstacle to axonal regeneration. The present study was designed to examine whether cell transplantation into a damaged central nervous system can reduce fibrotic scar formation and promote axonal regeneration. Nigrostriatal dopaminergic axons were unilaterally transected in rats and cultures of olfactory-ensheathing cells (OECs), and olfactory nerve fibroblasts were transplanted into the lesion site. In the absence of transplants, few tyrosine hydroxylase-immunoreactive axons extended across the lesion 2 weeks after the transection. Reactive astrocytes increased around the lesion, and a fibrotic scar containing type IV collagen deposits developed in the lesion center. The immunoreactivity of chondroitin sulfate side chains and core protein of NG2 proteoglycan increased in and around the lesion. One and 2 weeks after transection and simultaneous transplantation, dopaminergic axons regenerated across the transplanted tissues, which consisted of p75-immunoreactive OECs and fibronectin-immunoreactive fibroblasts. Reactive astrocytes and chondroitin sulfate immunoreactivity increased around the transplants, whereas the deposition of type IV collagen and fibrotic scar formation were completely prevented at the lesion site. Transplantation of meningeal fibroblasts similarly prevented the formation of the fibrotic scar, although its effect on regeneration was less potent than transplantation of OECs and olfactory nerve fibroblasts. The present results suggest that elimination of the inhibitory fibrotic scar is important for neural regeneration.

  4. Telomerase protects adult rodent olfactory ensheathing glia from early senescence.

    PubMed

    Llamusí, María-Beatriz; Rubio, Mari-Paz; Ramón-Cueto, Almudena

    2011-05-01

    Adult olfactory bulb ensheathing glia (OB-OEG) promote the repair of acute, subacute, and chronic spinal cord injuries and autologous transplantation is a feasible approach. There are interspecies differences between adult rodent and primate OB-OEG related to their longevity in culture. Whereas primate OB-OEG exhibit a relatively long life span, under the same culture conditions rodent OB-OEG divide just three to four times, are sensitive to oxidative stress and become senescent after the third week in vitro. Telomerase is a "physiological key regulator" of the life span of normal somatic cells and also has extratelomeric functions such as increased resistance to oxidative stress. To elucidate whether telomerase has a role in the senescence of rodent OB-OEG, we have introduced the catalytic subunit of telomerase mTERT into cultures of these cells by retroviral infection. Native and modified adult rat OB-OEG behaved as telomerase-competent cells as they divided while expressing mTERT but entered senescence once the gene switched off. After ectopic expression of mTERT, OB-OEG resumed division at a nonsenescent rate, expressed p75 and other OEG markers, and exhibited the morphology of nonsenescent OB-OEG. The nonsenescent period of mTERT-OEG lasted 9weeks and then ectopic mTERT switched off and cells entered senescence again. Our results suggest a role of telomerase in early senescence of adult rodent OB-OEG cultures and a protection from oxidative damage. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.

  5. Sciatic nerve repair with tissue engineered nerve: Olfactory ensheathing cells seeded poly(lactic-co-glygolic acid) conduit in an animal model

    PubMed Central

    Tan, C W; Ng, M H; Ohnmar, H; Lokanathan, Y; Nur-Hidayah, H; Roohi, S A; Ruszymah, BHI; Nor-Hazla, M H; Shalimar, A; Naicker, A S

    2013-01-01

    Background and Aim: Synthetic nerve conduits have been sought for repair of nerve defects as the autologous nerve grafts causes donor site morbidity and possess other drawbacks. Many strategies have been investigated to improve nerve regeneration through synthetic nerve guided conduits. Olfactory ensheathing cells (OECs) that share both Schwann cell and astrocytic characteristics have been shown to promote axonal regeneration after transplantation. The present study was driven by the hypothesis that tissue-engineered poly(lactic-co-glycolic acid) (PLGA) seeded with OECs would improve peripheral nerve regeneration in a long sciatic nerve defect. Materials and Methods: Sciatic nerve gap of 15 mm was created in six adult female Sprague-Dawley rats and implanted with PLGA seeded with OECs. The nerve regeneration was assessed electrophysiologically at 2, 4 and 6 weeks following implantation. Histopathological examination, scanning electron microscopic (SEM) examination and immunohistochemical analysis were performed at the end of the study. Results: Nerve conduction studies revealed a significant improvement of nerve conduction velocities whereby the mean nerve conduction velocity increases from 4.2 ΁ 0.4 m/s at week 2 to 27.3 ΁ 5.7 m/s at week 6 post-implantation (P < 0.0001). Histological analysis revealed presence of spindle-shaped cells. Immunohistochemical analysis further demonstrated the expression of S100 protein in both cell nucleus and the cytoplasm in these cells, hence confirming their Schwann-cell-like property. Under SEM, these cells were found to be actively secreting extracellular matrix. Conclusion: Tissue-engineered PLGA conduit seeded with OECs provided a permissive environment to facilitate nerve regeneration in a small animal model. PMID:24379458

  6. Axonal Ensheathment and Intercellular Barrier Formation in Drosophila

    PubMed Central

    Blauth, Kevin; Banerjee, Swati; Bhat, Manzoor A.

    2014-01-01

    Glial cells are critical players in every major aspect of nervous system development, function, and disease. Other than their traditional supportive role, glial cells perform a variety of important functions such as myelination, synapse formation and plasticity, and establishment of blood–brain and blood–nerve barriers in the nervous system. Recent studies highlight the striking functional similarities between Drosophila and vertebrate glia. In both systems, glial cells play an essential role in neural ensheathment thereby isolating the nervous system and help to create a local ionic microenvironment for conduction of nerve impulses. Here, we review the anatomical aspects and the molecular players that underlie ensheathment during different stages of nervous system development in Drosophila and how these processes lead to the organization of neuroglial junctions. We also discuss some key aspects of the invertebrate axonal ensheathment and junctional organization with that of vertebrate myelination and axon–glial interactions. Finally, we highlight the importance of intercellular junctions in barrier formation in various cellular contexts in Drosophila. We speculate that unraveling the genetic and molecular mechanisms of ensheathment across species might provide key insights into human myelin-related disorders and help in designing therapeutic interventions. PMID:20801419

  7. A novel method using intranasal delivery of EdU demonstrates that accessory olfactory ensheathing cells respond to injury by proliferation.

    PubMed

    Chehrehasa, Fatemeh; Ekberg, Jenny A K; St John, James A

    2014-03-20

    Olfactory ensheathing cells (OECs) play an important role in the continuous regeneration of the primary olfactory nervous system throughout life and for regeneration of olfactory neurons after injury. While it is known that several individual OEC subpopulations with distinct properties exist in different anatomical locations, it remains unclear how these different subpopulations respond to a major injury. We have examined the proliferation of OECs from one distinct location, the peripheral accessory olfactory nervous system, following large-scale injury (bulbectomy) in mice. We used crosses of two transgenic reporter mouse lines, S100ß-DsRed and OMP-ZsGreen, to visualise OECs, and main/accessory olfactory neurons, respectively. We surgically removed one olfactory bulb including the accessory olfactory bulb to induce degeneration, and found that accessory OECs in the nerve bundles that terminate in the accessory olfactory bulb responded by increased proliferation with a peak occurring 2 days after the injury. To label proliferating cells we used the thymidine analogue ethynyl deoxyuridine (EdU) using intranasal delivery instead of intraperitoneal injection. We compared and quantified the number of proliferating cells at different regions at one and four days after EdU labelling by the two different methods and found that intranasal delivery method was as effective as intraperitoneal injection. We demonstrated that accessory OECs actively respond to widespread degeneration of accessory olfactory axons by proliferating. These results have important implications for selecting the source of OECs for neural regeneration therapies and show that intranasal delivery of EdU is an efficient and reliable method for assessing proliferation of olfactory glia.

  8. Burkholderia pseudomallei Capsule Exacerbates Respiratory Melioidosis but Does Not Afford Protection against Antimicrobial Signaling or Bacterial Killing in Human Olfactory Ensheathing Cells

    PubMed Central

    Dando, Samantha J.; Ipe, Deepak S.; Batzloff, Michael; Sullivan, Matthew J.; Crossman, David K.; Crowley, Michael; Strong, Emily; Kyan, Stephanie; Leclercq, Sophie Y.; Ekberg, Jenny A. K.; St. John, James

    2016-01-01

    Melioidosis, caused by the bacterium Burkholderia pseudomallei, is an often severe infection that regularly involves respiratory disease following inhalation exposure. Intranasal (i.n.) inoculation of mice represents an experimental approach used to study the contributions of bacterial capsular polysaccharide I (CPS I) to virulence during acute disease. We used aerosol delivery of B. pseudomallei to establish respiratory infection in mice and studied CPS I in the context of innate immune responses. CPS I improved B. pseudomallei survival in vivo and triggered multiple cytokine responses, neutrophil infiltration, and acute inflammatory histopathology in the spleen, liver, nasal-associated lymphoid tissue, and olfactory mucosa (OM). To further explore the role of the OM response to B. pseudomallei infection, we infected human olfactory ensheathing cells (OECs) in vitro and measured bacterial invasion and the cytokine responses induced following infection. Human OECs killed >90% of the B. pseudomallei in a CPS I-independent manner and exhibited an antibacterial cytokine response comprising granulocyte colony-stimulating factor, tumor necrosis factor alpha, and several regulatory cytokines. In-depth genome-wide transcriptomic profiling of the OEC response by RNA-Seq revealed a network of signaling pathways activated in OECs following infection involving a novel group of 378 genes that encode biological pathways controlling cellular movement, inflammation, immunological disease, and molecular transport. This represents the first antimicrobial program to be described in human OECs and establishes the extensive transcriptional defense network accessible in these cells. Collectively, these findings show a role for CPS I in B. pseudomallei survival in vivo following inhalation infection and the antibacterial signaling network that exists in human OM and OECs. PMID:27091931

  9. Olfactory Ensheathing Cell Transplantation in Experimental Spinal Cord Injury: Effect size and Reporting Bias of 62 Experimental Treatments: A Systematic Review and Meta-Analysis

    PubMed Central

    Sena, Emily S.; Dirnagl, Ulrich

    2016-01-01

    Olfactory ensheathing cell (OEC) transplantation is a candidate cellular treatment approach for human spinal cord injury (SCI) due to their unique regenerative potential and autologous origin. The objective of this study was, through a meta-epidemiologic approach, (i) to assess the efficacy of OEC transplantation on locomotor recovery after traumatic experimental SCI and (ii) to estimate the likelihood of reporting bias and/or missing data. A study protocol was finalized before data collection. Embedded into a systematic review and meta-analysis, we conducted a literature research of databases including PubMed, EMBASE, and ISI Web of Science from 1949/01 to 2014/10 with no language restrictions, screened by two independent investigators. Studies were included if they assessed neurobehavioral improvement after traumatic experimental SCI, administrated no combined interventions, and reported the number of animals in the treatment and control group. Individual effect sizes were pooled using a random effects model. Details regarding the study design were extracted and impact of these on locomotor outcome was assessed by meta-regression. Missing data (reporting bias) was determined by Egger regression and Funnel-plotting. The primary study outcome assessed was improvement in locomotor function at the final time point of measurement. We included 49 studies (62 experiments, 1,164 animals) in the final analysis. The overall improvement in locomotor function after OEC transplantation, measured using the Basso, Beattie, and Bresnahan (BBB) score, was 20.3% (95% CI 17.8–29.5). One missing study was imputed by trim and fill analysis, suggesting only slight publication bias and reducing the overall effect to a 19.2% improvement of locomotor activity. Dose-response ratio supports neurobiological plausibility. Studies were assessed using a 9-point item quality score, resulting in a median score of 5 (interquartile range [IQR] 3–5). In conclusion, OEC transplantation exerts

  10. TNFa/TNFR2 signaling is required for glial ensheathment at the dorsal root entry zone.

    PubMed

    Smith, Cody J; Wheeler, Michael A; Marjoram, Lindsay; Bagnat, Michel; Deppmann, Christopher D; Kucenas, Sarah

    2017-04-05

    Somatosensory information from the periphery is routed to the spinal cord through centrally-projecting sensory axons that cross into the central nervous system (CNS) via the dorsal root entry zone (DREZ). The glial cells that ensheath these axons ensure rapid propagation of this information. Despite the importance of this glial-axon arrangement, how this afferent nerve is assembled during development is unknown. Using in vivo, time-lapse imaging we show that as centrally-projecting pioneer axons from dorsal root ganglia (DRG) enter the spinal cord, they initiate expression of the cytokine TNFalpha. This induction coincides with ensheathment of these axons by associated glia via a TNF receptor 2 (TNFR2)-mediated process. This work identifies a signaling cascade that mediates peripheral glial-axon interactions and it functions to ensure that DRG afferent projections are ensheathed after pioneer axons complete their navigation, which promotes efficient somatosensory neural function.

  11. CNS-derived glia ensheath peripheral nerves and mediate motor root development.

    PubMed

    Kucenas, Sarah; Takada, Norio; Park, Hae-Chul; Woodruff, Elvin; Broadie, Kendal; Appel, Bruce

    2008-02-01

    Motor function requires that motor axons extend from the spinal cord at regular intervals and that they are myelinated by Schwann cells. Little attention has been given to another cellular structure, the perineurium, which ensheaths the motor nerve, forming a flexible, protective barrier. Consequently, the origin of perineurial cells and their roles in motor nerve formation are poorly understood. Using time-lapse imaging in zebrafish, we show that perineurial cells are born in the CNS, arising as ventral spinal-cord glia before migrating into the periphery. In embryos lacking perineurial glia, motor neurons inappropriately migrated outside of the spinal cord and had aberrant axonal projections, indicating that perineurial glia carry out barrier and guidance functions at motor axon exit points. Additionally, reciprocal signaling between perineurial glia and Schwann cells was necessary for motor nerve ensheathment by both cell types. These insights reveal a new class of CNS-born glia that critically contributes to motor nerve development.

  12. Immunocytochemical characterisation of ensheathing glia in the olfactory and vomeronasal systems of Ambystoma mexicanum (Caudata: Ambystomatidae).

    PubMed

    Lazzari, Maurizio; Bettini, Simone; Franceschini, Valeria

    2016-03-01

    The olfactory and vomeronasal systems of vertebrates are characterised by neurogenesis occurring throughout life. The regenerative ability of olfactory receptor neurons relies on specific glial cells, the olfactory and vomeronasal axon-surrounding cells. Numerous studies have examined mammalian olfactory ensheathing cells which are considered potential candidates for spinal cord injury repair using cell-based therapy. With regard to non-mammalian vertebrates, limited information is available on these glial cells in fish, and there is no information on them in terrestrial anamniotes, the amphibians. In the present research, we studied the immunocytochemical characteristics of axon-surrounding cells in Ambystoma mexicanum. Urodeles have relatively simple olfactory and vomeronasal systems, and represent a good model for studying ensheathing cells in extant representatives of basal tetrapods. Sections from the decalcified heads of A. mexicanum were immunocytochemically processed for the detection of proteins used in research on mammalian olfactory-ensheathing cells. S100, GFAP and NCAM were clearly observed. p75NTR, Gal-1 and PSA-NCAM showed weak staining. No vimentin immunopositivity was observed. The corresponding areas of the olfactory and vomeronasal pathways displayed the same staining characteristics, with the exception of Gal-1, p75NTR and PSA-NCAM in the mucosae. The degree of marker expression was not uniform throughout the sensory pathways. In contrast to fish, both olfactory and vomeronasal nerves displayed uniform staining intensity. This study showed that some markers for mammalian and fish-ensheathing glia are also applicable in urodeles. The olfactory systems of vertebrates show similarities, and also clear dissimilarities. Further investigations are required to ascertain the functional significance of these regional and interspecific differences.

  13. A large fraction of neocortical myelin ensheathes axons of local inhibitory neurons

    PubMed Central

    Micheva, Kristina D; Wolman, Dylan; Mensh, Brett D; Pax, Elizabeth; Buchanan, JoAnn; Smith, Stephen J; Bock, Davi D

    2016-01-01

    Myelin is best known for its role in increasing the conduction velocity and metabolic efficiency of long-range excitatory axons. Accordingly, the myelin observed in neocortical gray matter is thought to mostly ensheath excitatory axons connecting to subcortical regions and distant cortical areas. Using independent analyses of light and electron microscopy data from mouse neocortex, we show that a surprisingly large fraction of cortical myelin (half the myelin in layer 2/3 and a quarter in layer 4) ensheathes axons of inhibitory neurons, specifically of parvalbumin-positive basket cells. This myelin differs significantly from that of excitatory axons in distribution and protein composition. Myelin on inhibitory axons is unlikely to meaningfully hasten the arrival of spikes at their pre-synaptic terminals, due to the patchy distribution and short path-lengths observed. Our results thus highlight the need for exploring alternative roles for myelin in neocortical circuits. DOI: http://dx.doi.org/10.7554/eLife.15784.001 PMID:27383052

  14. How Cells Endure Extreme Conditions

    SciTech Connect

    2009-01-01

    One of natures most gripping feats of survival is now better understood. For the first time, Berkeley Lab scientists observed the chemical changes in individual cells that enable them to survive in conditions that should kill them. http://newscenter.lbl.gov/feature-stories/2009/07/07/cells-endure-extremes/

  15. Evidence that contact with connective tissue matrix is required for normal interaction between Schwann cells and nerve fibers

    PubMed Central

    1978-01-01

    Explants of fetal rat sensory ganglia, cultured under conditions allowing axon and Schwann cell outgrowth in the absence of fibroblasts, occasionally develop nerve fascicles that are partially suspended in culture medium above the collagen substrate. In these suspended regions, fascicles are abnormal in that Schwann cells are decreased in number, are confined to occasional clusters along the fascicle, provide ensheathment for only a few axons at the fascicle periphery, and do not form myelin. When these fascicles are presented with a substrate of reconstituted rat-tail collagen, Schwann cell numbers increase, ensheathment of small nerve fibers occurs normally, and larger axons are myelinated. We conclude that, for normal development, Schwann cells require contact with extracellular matrix as well as axons. The Schwann cell abnormalities in suspended fascicles are similar to those observed in nerve roots of dystrophic mice. PMID:701366

  16. Fray, a Drosophila serine/threonine kinase homologous to mammalian PASK, is required for axonal ensheathment

    NASA Technical Reports Server (NTRS)

    Leiserson, W. M.; Harkins, E. W.; Keshishian, H.

    2000-01-01

    Fray is a serine/threonine kinase expressed by the peripheral glia of Drosophila, whose function is required for normal axonal ensheathment. Null fray mutants die early in larval development and have nerves with severe swelling and axonal defasciculation. The phenotype is associated with a failure of the ensheathing glia to correctly wrap peripheral axons. When the fray cDNA is expressed in the ensheathing glia of fray mutants, normal nerve morphology is restored. Fray belongs to a novel family of Ser/Thr kinases, the PF kinases, whose closest relatives are the PAK kinases. Rescue of the Drosophila mutant phenotype with PASK, the rat homolog of Fray, demonstrates a functional homology among these proteins and suggests that the Fray signaling pathway is widely conserved.

  17. Fuel-cell engine stream conditioning system

    DOEpatents

    DuBose, Ronald Arthur

    2002-01-01

    A stream conditioning system for a fuel cell gas management system or fuel cell engine. The stream conditioning system manages species potential in at least one fuel cell reactant stream. A species transfer device is located in the path of at least one reactant stream of a fuel cell's inlet or outlet, which transfer device conditions that stream to improve the efficiency of the fuel cell. The species transfer device incorporates an exchange media and a sorbent. The fuel cell gas management system can include a cathode loop with the stream conditioning system transferring latent and sensible heat from an exhaust stream to the cathode inlet stream of the fuel cell; an anode humidity retention system for maintaining the total enthalpy of the anode stream exiting the fuel cell related to the total enthalpy of the anode inlet stream; and a cooling water management system having segregated deionized water and cooling water loops interconnected by means of a brazed plate heat exchanger.

  18. Sourcing different neuro-progenitor cell for the use of nerve construct.

    PubMed

    Yazid, A G M; Anuar, A; Onhmar, H T W E; Ng, A M H; Ruszymah, B H I; Amaramalar, S N

    2008-07-01

    Spinal cord, sciatic nerve, olfactory ensheathing cell and bone marrow derived mesenchymal stem cells were evaluated as an alternative source for tissue engineering of nerve conduit. All cell sources were cultured in alpha-MEM medium. Olfactory Ensheathing Cell (OEC) showed the best result with higher growth kinetic compared to the others. Spinal cord and sciatic nerve were positive for GFAP, OEC were positive for GFAP, S100b and anti-cytokeratin 18 but negative for anti-Human Fibroblast.

  19. Methods of conditioning direct methanol fuel cells

    DOEpatents

    Rice, Cynthia; Ren, Xiaoming; Gottesfeld, Shimshon

    2005-11-08

    Methods for conditioning the membrane electrode assembly of a direct methanol fuel cell ("DMFC") are disclosed. In a first method, an electrical current of polarity opposite to that used in a functioning direct methanol fuel cell is passed through the anode surface of the membrane electrode assembly. In a second method, methanol is supplied to an anode surface of the membrane electrode assembly, allowed to cross over the polymer electrolyte membrane of the membrane electrode assembly to a cathode surface of the membrane electrode assembly, and an electrical current of polarity opposite to that in a functioning direct methanol fuel cell is drawn through the membrane electrode assembly, wherein methanol is oxidized at the cathode surface of the membrane electrode assembly while the catalyst on the anode surface is reduced. Surface oxides on the direct methanol fuel cell anode catalyst of the membrane electrode assembly are thereby reduced.

  20. Axon Regeneration Can Facilitate or Suppress Hindlimb Function after Olfactory Ensheathing Glia Transplantation

    PubMed Central

    Takeoka, Aya; Jindrich, Devin L.; Muñoz-Quiles, Cintia; Zhong, Hui; van den Brand, Rubia; Pham, Daniel L.; Ziegler, Matthias D.; Ramón-Cueto, Almudena; Roy, Roland R.; Edgerton, V. Reggie

    2011-01-01

    Reports based primarily on anatomical evidence suggest that olfactory ensheathing glia (OEG) transplantation promotes axon regeneration across a complete spinal cord transection in adult rats. Based on functional, electrophysiological, and anatomical assessments, we found that OEG promoted axon regeneration across a complete spinal cord transection and that this regeneration altered motor responses over time. At 7 months after transection, 70% of OEG-treated rats showed motor-evoked potentials in hindlimb muscles after transcranial electric stimulation. Furthermore, a complete spinal cord retransection performed 8 months after injury demonstrated that this axon regeneration suppressed locomotor performance and decreased the hypersensitive hindlimb withdrawal response to mechanical stimulation. OEG transplantation alone promoted reorganization of lumbosacral locomotor networks and, when combined with long-term training, enhanced some stepping measures. These novel findings demonstrate that OEG promote regeneration of mature axons across a complete transection and reorganization of spinal circuitry, both of which contribute to sensorimotor function. PMID:21411671

  1. Stem cell therapy in spinal cord injury: Hollow promise or promising science?

    PubMed Central

    Goel, Aimee

    2016-01-01

    Spinal cord injury (SCI) remains one of the most physically, psychologically and socially debilitating conditions worldwide. While rehabilitation measures may help limit disability to some extent, there is no effective primary treatment yet available. The efficacy of stem cells as a primary therapeutic option in spinal cord injury is currently an area under much scrutiny and debate. Several laboratory and some primary clinical studies into the use of bone marrow mesenchymal stem cells or embryonic stem cell-derived oligodentrocyte precursor cells have shown some promising results in terms of remyelination and regeneration of damaged spinal nerve tracts. More recently,laboratory and early clinical experiments into the use of Olfactory Ensheathing Cells, a type of glial cell derived from olfactory bulb and mucosa have provided some phenomenal preliminary evidence as to their neuroregenerative and neural bridging capacity. This report compares and evaluates some current research into selected forms of embryonic and mesenchymal stem cell therapy as well as olfactory ensheathing cell therapy in SCI, and also highlights some legal and ethical issues surrounding their use. While early results shows promise, more rigorous large scaleclinical trials are needed to shed light on the safety, efficacy and long term viability of stem cell and cellular transplant techniques in SCI. PMID:27217662

  2. Improved conditions for murine epidermal cell culture.

    PubMed

    Fischer, S M; Viaje, A; Harris, K L; Miller, D R; Bohrman, J S; Slaga, T J

    1980-02-01

    An improved method for cultivating newborn mouse epidermal cells has been developed that increases the longevity, epithelial nature and efficiency of cell-line establishment. The use of Super Medium, an enriched Waymouth's formulation, increased proliferation for long periods of time, as did incubation at 31 degrees C rather than 37 degrees C. The fetal bovine serum requirement was found to be reduced at the lower temperature. An increase in labeling indices was seen when epidermal growth factor (EGF) or the cyclic nucleotides were added and the presence of EGF receptors was determined. Of the prostaglandins (PG) examined, PGE1 and PGE2 produced the greatest increase in DNA synthesis. The PG precursors, arachidonic and 8,11,14-eicosatrienoic acid, were also greatly stimulatory. The use of a lethally irradiated 3T3 feeder layer at 31 degrees C proved superior in maintenance of an epithelial morphology. Subculturable cell lines were established much more readily and reproducibly in carcinogen-treated cultures grown under the improved conditions.

  3. Chronic Spinal Injury Repair by Olfactory Bulb Ensheathing Glia and Feasibility for Autologous Therapy

    PubMed Central

    Muñoz-Quiles, Cintia; Santos-Benito, Fernando F.; Llamusí, M. Beatriz; Ramón-Cueto, Almudena

    2009-01-01

    Olfactory bulb ensheathing glia (OB-OEG) promote repair of spinal cord injury (SCI) in rats after transplantation at acute or subacute (up to 45 days) stages. The most relevant clinical scenario in humans, however, is chronic SCI, in which no more major cellular or molecular changes occur at the injury site; this occurs after the third month in rodents. Whether adult OB-OEG grafts promote repair of severe chronic SCI has not been previously addressed. Rats with complete SCI that were transplanted with OB-OEG 4 months after injury exhibited progressive improvement in motor function and axonal regeneration from different brainstem nuclei across and beyond the SCI site. A positive correlation between motor outcome and axonal regeneration suggested a role for brainstem neurons in the recovery. Functional and histological outcomes did not differ at subacute or chronic stages. Thus, autologous transplantation is a feasible approach as there is time for patient stabilization and OEG preparation in human chronic SCI; the healing effects of OB-OEG on established injuries may offer new therapeutic opportunities for chronic SCI patients. PMID:19915486

  4. Sertoli cell condition medium can induce germ like cells from bone marrow derived mesenchymal stem cells

    PubMed Central

    Monfared, Mahdieh Hajian; Minaee, Bagher; Rastegar, Tayebeh; Khrazinejad, Ebrahim; Barbarestani, Mohammad

    2016-01-01

    Objective(s): Although many researchers have confirmed induction of germ cells from bone marrow mesenchymal stem cells (BMMSCs), there are no reports that confirm spontaneous differentiation of germ cells from BMMSCs. In this study, we have evaluated the effect of adult Sertoli cell condition medium (SCCM) as a mutative factor in the induction of germ cells from BMMSCs. Materials and Methods: BMMSCs were collected from the bone marrow of 6-8-week old NMRI mice and their mesenchymal entities were proven using superficial markers (expression of CD44 and CD73 and non-expresion of CD45 and CD11b) by fow cytometry. Their multi-potential entities were proved with differentiation to osteogenic and adipogenic cells for 21 days. Also isolated Sertoli cells were enriched using lectin coated plates and Sertoli cell condition medium (SCCM) was collected. Sertoli cells were identified by immunocytochemistry and Vimentin marker. The cells were then differentiated into germ cells with SCCM for 2 weeks. Finally induced cells were evaluated by RT-PCR and immunocytochemistry. Results: Differentiation of mesenchymal stem cells to osteoblast and adipocyte showed their multi-potential property. Expression of CD44 and CD73 and non-expression of CD45 and CD11b confirmed mesenchyme cells. Immunocytochemistry and RT-PCR results showed expression of germ cells specific marker (Mvh). Conclusion: This study confirmed the effect of SCCM as a motivational factor that can used for differentiation of germ cells from BMMSCs. PMID:27917274

  5. An update on Schwann cell biology--immunomodulation, neural regulation and other surprises.

    PubMed

    Armati, Patricia J; Mathey, Emily K

    2013-10-15

    Schwann cells are primarily discussed in the context of their ability to form myelin. However there are many subtypes of these neural crest derived cells including satellite cells of the dorsal root ganglia and autonomic ganglia, the perisynaptic Schwann cells of the neuromuscular junction and the non-myelin forming Schwann cells which ensheathe the unmyelinated fibres of the peripheral nervous system which are about 80% of peripheral nerves. This review discusses the many functions of these Schwann cell subsets including their seminal role in axonal ensheathment, perineuronal organisation, maintenance of normal neural function, synapse formation, response to damage and repair and an increasingly recognised active role in pain syndromes.

  6. Olfactory ensheathing glia: their contribution to primary olfactory nervous system regeneration and their regenerative potential following transplantation into the injured spinal cord.

    PubMed

    Franssen, Elske H P; de Bree, Freddy M; Verhaagen, Joost

    2007-11-01

    Olfactory ensheathing glia (OEG) are a specialized type of glia that guide primary olfactory axons from the neuroepithelium in the nasal cavity to the brain. The primary olfactory system is able to regenerate after a lesion and OEG contribute to this process by providing a growth-supportive environment for newly formed axons. In the spinal cord, axons are not able to restore connections after an injury. The effects of OEG transplants on the regeneration of the injured spinal cord have been studied for over a decade. To date, of all the studies using only OEG as a transplant, 41 showed positive effects, while 13 studies showed limited or no effects. There are several contradictory reports on the migratory and axon growth-supporting properties of transplanted OEG. Hence, the regenerative potential of OEG has become the subject of intense discussion. In this review, we first provide an overview of the molecular and cellular characteristics of OEG in their natural environment, the primary olfactory nervous system. Second, their potential to stimulate regeneration in the injured spinal cord is discussed. OEG influence scar formation by their ability to interact with astrocytes, they are able to remyelinate axons and promote angiogenesis. The ability of OEG to interact with scar tissue cells is an important difference with Schwann cells and may be a unique characteristic of OEG. Because of these effects after transplantation and because of their role in primary olfactory system regeneration, the OEG can be considered as a source of neuroregeneration-promoting molecules. To identify these molecules, more insight into the molecular biology of OEG is required. We believe that genome-wide gene expression studies of OEG in their native environment, in culture and after transplantation will ultimately reveal unique combinations of molecules involved in the regeneration-promoting potential of OEG.

  7. Test facility for solar-cell reference conditions

    NASA Technical Reports Server (NTRS)

    Klucher, T. M.

    1976-01-01

    A test facility, intended primarily for long-term monitoring of the global insolation and its components and the concurrent solar cell performance under a wide variety of measureable atmospheric and weather conditions, is described. Instruments for the measurement of insolation, cell performance, turbidity, water vapor, and cloud cover are described. Preliminary evaluation of the hourly data base generated over a two-month period for a range of sky conditions from clear to overcast is presented.

  8. Regeneration of the Adult Rat Spinal Cord in Response to Ensheathing Cells and Methylprednisolone

    DTIC Science & Technology

    2002-01-01

    me in academics and research, and also as my friend. I thank Dr. Linda L. Porter, for her continuous efforts on my behalf as the Chairperson of...and Spinal Cord Injury Program. We are grateful to Drs. Barbara S. Bregman and Linda L. Porter for their wonderful suggestions and guidance; to Anna...ES, Pietronigro DD, Seligman ML (1980) The free radical pathology and the microcirculation in the major central nervous system disorders. Acta

  9. [Pathogenesis of spinal cord injuries and mechanisms of repair induced by olfactory ensheathing cells].

    PubMed

    Botero, Lucía; Gomez, Rosa Margarita; Chaparro, Orlando

    2013-05-16

    Introduccion. La lesion medular es un evento catastrofico, cuyas consecuencias persisten durante toda la vida del paciente. La investigacion en tratamiento se ha basado principalmente en el desarrollo de terapias que reduzcan la discapacidad, pero desde los anos noventa hay un avance significativo y se han probado varios trasplantes celulares en modelos animales de lesion medular, celulas de Schwann, astrocitos y celulas de la glia envolvente olfatoria (CGEO). Objetivo. Hacer un recuento detallado de la patogenia de la lesion medular primaria y secundaria y de los mecanismos por los cuales las CGEO inducirian sus posibles efectos regenerativos descritos en la bibliografia. Desarrollo. Despues del traumatismo, la lesion se desarrolla en dos fases, la primaria se caracteriza por las lesiones de compresion y la secundaria se produce por una serie de factores que se dan en paralelo y que incluyen factores vasculares, celulares, moleculares y formacion de cicatriz glial. La mayoria de los modelos de lesion medular y trasplante con CGEO han comunicado recuperacion funcional, remielinizacion y regeneracion axonal. Estas celulas ejercen su accion de manera indirecta a traves de la produccion de factores de crecimiento y de manera directa induciendo regeneracion neuronal, axonal y remielinizacion. Conclusiones. Las CGEO son una opcion terapeutica en pacientes con lesion medular debido a que inducen de modo directo o indirecto regeneracion neuronal, axonal, remielinizacion de axones, disminucion de cicatriz glial y otros efectos que conducen a la recuperacion funcional.

  10. Condition number analysis and preconditioning of the finite cell method

    NASA Astrophysics Data System (ADS)

    de Prenter, F.; Verhoosel, C. V.; van Zwieten, G. J.; van Brummelen, E. H.

    2017-04-01

    The (Isogeometric) Finite Cell Method - in which a domain is immersed in a structured background mesh - suffers from conditioning problems when cells with small volume fractions occur. In this contribution, we establish a rigorous scaling relation between the condition number of (I)FCM system matrices and the smallest cell volume fraction. Ill-conditioning stems either from basis functions being small on cells with small volume fractions, or from basis functions being nearly linearly dependent on such cells. Based on these two sources of ill-conditioning, an algebraic preconditioning technique is developed, which is referred to as Symmetric Incomplete Permuted Inverse Cholesky (SIPIC). A detailed numerical investigation of the effectivity of the SIPIC preconditioner in improving (I)FCM condition numbers and in improving the convergence speed and accuracy of iterative solvers is presented for the Poisson problem and for two- and three-dimensional problems in linear elasticity, in which Nitche's method is applied in either the normal or tangential direction. The accuracy of the preconditioned iterative solver enables mesh convergence studies of the finite cell method.

  11. Hypoxic culture conditions enhance the generation of regulatory T cells

    PubMed Central

    Neildez-Nguyen, Thi My Anh; Bigot, Jérémy; Da Rocha, Sylvie; Corre, Guillaume; Boisgerault, Florence; Paldi, Andràs; Galy, Anne

    2015-01-01

    The generation of large amounts of induced CD4+ CD25+ Foxp3+ regulatory T (iTreg) cells is of great interest for several immunotherapy applications, therefore a better understanding of signals controlling iTreg cell differentiation and expansion is required. There is evidence that oxidative metabolism may regulate several key signalling pathways in T cells. This prompted us to investigate the effects of oxygenation on iTreg cell generation by comparing the effects of atmospheric (21%) or of low (5%) O2 concentrations on the phenotype of bead-stimulated murine splenic CD4+ T cells from Foxp3-KI-GFP T-cell receptor transgenic mice. The production of intracellular reactive oxygen species was shown to play a major role in the generation of iTreg cells, a process characterized by increased levels of Sirt1, PTEN and Glut1 on the committed cells, independently of the level of oxygenation. The suppressive function of iTreg cells generated either in atmospheric or low oxygen levels was equivalent. However, greater yields of iTreg cells were obtained under low oxygenation, resulting from a higher proliferative rate of the committed Treg cells and higher levels of Foxp3, suggesting a better stability of the differentiation process. Higher expression of Glut1 detected on iTreg cells generated under hypoxic culture conditions provides a likely explanation for the enhanced proliferation of these cells as compared to those cultured under ambient oxygen. Such results have important implications for understanding Treg cell homeostasis and developing in vitro protocols for the generation of Treg cells from naive T lymphocytes. PMID:25243909

  12. Optimization of conditions and cell feeding procedures for alcohol fermentation

    SciTech Connect

    Ciftci, T.; Constantinides, A.; Wang, S.S.

    1983-08-01

    Alcohol fermentation was studied with an emphasis on the separation of cell growth and alcohol production stages. Experiments were conducted to establish the optimal conditions for alcohol production in batch fermentations and to simulate continuous fermentations with cell feeding at various stages. It was found that the glucose concentration should be kept under 10% (w/v), and the temperature should be between 40 and 42.5 degrees C for maximum specific alcohol productivity. If the cell concentration is increased, a decrease in specific alcohol productivity is observed. Higher cell concentrations are needed for higher final alcohol concentrations. Among the cell feeding procedures into alcohol production stages, a cocurrent design was found to be better than recycle and countercurrent designs. (30 Refs.)

  13. FHL2 regulates hematopoietic stem cell functions under stress conditions

    PubMed Central

    Hou, Yu; Wang, Xiaoqin; Li, LiPing; Fan, Rong; Chen, Ju; Zhu, Tongyu; Li, Wen; Jiang, Yanwen; Mittal, Nupur; Wu, Wenshu; Peace, David; Qian, Zhijian

    2014-01-01

    FHL2, a member of the four and one half LIM domain protein family, is a critical transcriptional modulator. Here, we identify FHL2 as a critical regulator of hematopoietic stem cells (HSCs) that is essential for maintaining HSC self-renewal under regenerative stress. We find that Fhl2 loss has limited effects on hematopoiesis under homeostatic conditions. In contrast, Fhl2-null chimeric mice reconstituted with Fhl2-null bone marrow cells developed abnormal hematopoiesis with significantly reduced numbers of HSCs, hematopoietic progenitor cells (HPCs), red blood cells and platelets as well as hemoglobin levels. In addition, HSCs displayed a significantly reduced self-renewal capacity and were skewed toward myeloid lineage differentiation. We find that Fhl2 loss reduces both HSC quiescence and survival in response to regenerative stress, probably as a consequence of Fhl2-loss-mediated down-regulation of cyclin dependent kinase (CDK)-inhibitors, including p21(Cip) and p27(Kip1). Interestingly, FHL2 is regulated under control of a tissue specific promoter in hematopoietic cells and it is down-regulated by DNA hypermethylation in the leukemia cell line and primary leukemia cells. Furthermore, we find that down-regulation of FHL2 frequently occurs in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) patients, raising a possibility that FHL2 down-regulation plays a role in the pathogenesis of myeloid malignancies. PMID:25179730

  14. Conditions for Two-Cell Structure in Severe Vortical Storms.

    DTIC Science & Technology

    1984-02-01

    SEVERE VORTICAL STORMS by G. F. Carrier, F. E. Fendell , P. S. Feldman, and S. F. Fink TRW Space and Technology Group, Redondo Beach, CA 90278 Thi...Claification Conditions for Two-Call Structure in Severe Vortical Storms (U) 12. PERSONAL AUTHOR(S) Carrier. G. F. (Harvard U.): Fendell , F. E., Feldman...cell structure will occur. Very roughly, about half of all tropical storms ( Fendell 1974), and about one-quarter to one-half of meso- cyclones (Brooks

  15. Testing hot cell shielding in the fuel conditioning facility.

    PubMed

    Courtney, J C; Klann, R T

    1997-01-01

    A comprehensive shield test program for a hot cell complex, the Fuel Conditioning Facility at Argonne National Laboratory, has been completed with minimum radiation exposure to participants. The recently modified shielding design for two hot cells and their associated transfer paths for irradiated materials was analyzed and tested for attenuating gamma rays from mixed fission product sources. Testing was accomplished using 0.37 TBq (10 Ci) and 518 TBq (14,000 Ci) 60Co sources. Of specific concern were radiation levels around wall penetrations and the interface between transport casks and the cell floor. Detailed measurements were made for surfaces that bound the hot cells, a transfer tunnel between the two cells, and storage pits that extend below the floors of both cells. In addition to surface measurements, dose equivalent rates in adjacent corridors were determined when the larger source was exposed. Results indicate that with some administrative controls, the facility shields are adequate to meet the design criterion that limits annual dose to less than 10 mSv (1 rem) for facility workers.

  16. Efficient cultivation conditions for human limbal epithelial cells.

    PubMed

    Kim, Mee Kum; Lee, Jae Lim; Oh, Joo Youn; Shin, Mi Sun; Shin, Kyeong Seon; Wee, Won Ryang; Lee, Jin Hak; Park, Ki Sook; Son, Young Sook

    2008-10-01

    To compare the stem niche in different culture conditions of limbal epithelial cells, the suspended human limbal epithelial cells (HLECs) were seeded on the 3T3-pretreated plates and the other suspended cells were plated on amniotic membranes (AMs) which were either cryo-preserved or freeze-dried. All were cultured for 10 to 12 days. Reverse transcription-polymerase chain reaction (RT-PCR) for ATP-binding cassette, subfamily G, member 2 (ABCG2), p63, cytokeratin 12, and connexin 43 were performed in cultivated HLECs and their expression levels were compared. The mRNA expression of all markers examined showed no statistically significant differences between the cells on cryo-preserved and on freeze-dried AM. The expression of p63 and cytokeratin 12 in cultivated cells on AMs were significantly lower than those in 3T3-cocultured cells on RT-PCR and immunofluorescent staining. Cultivated HLECs on AMs showed reduced proliferation and differentiation while maintaining stem-property regardless of the preservative method of AM.

  17. Effects of ambient conditions on fuel cell vehicle performance

    NASA Astrophysics Data System (ADS)

    Haraldsson, K.; Alvfors, P.

    Ambient conditions have considerable impact on the performance of fuel cell hybrid vehicles. Here, the vehicle fuel consumption, the air compressor power demand, the water management system and the heat loads of a fuel cell hybrid sport utility vehicle (SUV) were studied. The simulation results show that the vehicle fuel consumption increases with 10% when the altitude increases from 0 m up to 3000 m to 4.1 L gasoline equivalents/100 km over the New European Drive Cycle (NEDC). The increase is 19% on the more power demanding highway US06 cycle. The air compressor is the major contributor to this fuel consumption increase. Its load-following strategy makes its power demand increase with increasing altitude. Almost 40% of the net power output of the fuel cell system is consumed by the air compressor at the altitude of 3000 m with this load-following strategy and is thus more apparent in the high-power US06 cycle. Changes in ambient air temperature and relative humidity effect on the fuel cell system performance in terms of the water management rather in vehicle fuel consumption. Ambient air temperature and relative humidity have some impact on the vehicle performance mostly seen in the heat and water management of the fuel cell system. While the heat loads of the fuel cell system components vary significantly with increasing ambient temperature, the relative humidity did not have a great impact on the water balance. Overall, dimensioning the compressor and other system components to meet the fuel cell system requirements at the minimum and maximum expected ambient temperatures, in this case 5 and 40 °C, and high altitude, while simultaneously choosing a correct control strategy are important parameters for efficient vehicle power train management.

  18. Multiscale molecular simulations of proteins in cell-like conditions

    NASA Astrophysics Data System (ADS)

    Samiotakis, Antonios

    Proteins are the workhorses of all living organisms, performing a broad range of functions in the crowded cellular interior. However, little is known about how proteins function in cell-like conditions since most studies focus in dilute aqueous environments. In order to address this problem we incorporated molecular simulations and coarse-grained models that capture the protein dynamics in the cellular interior. We study the macromolecular crowding effects of cell-like environments on protein Borrelia Burgdorferi VlsE (variable major protein-like sequence-expressed), an aspherical membrane protein, and the enzyme Phosphoglycerate kinase. We show that protein conformation can be significantly perturbed under crowded cell-like conditions which, in turn, can have dramatic effects to the proteins' function. In addition, we look into the effects of mutations in the folding pathways of the topologically frustrated protein apoflavodoxin while correlation with experiments is also achieved. We further developed a multiscale simulation scheme that combines the sampling efficiency of low-resolution models with the detail of all-atomistic simulations. An algorithm that reconstructs all-atomistic conformations from coarse-grained representations was developed, in addition to an energy function that accounts for chemical interference based on the Boltzamn inversion method. The multiscale simulation scheme manages to sample all-atomistic structures of the protein Trp-cage that match very well with experiments. The folding kinetic behavior of Trp-cage was also studied in the combined presence of urea denaturant and macromolecular crowding.

  19. Conditional telomerase induction causes proliferation of hair follicle stem cells

    PubMed Central

    Sarin, Kavita Y.; Cheung, Peggie; Gilison, Daniel; Lee, Eunice; Tennen, Ruth I.; Wang, Estee; Artandi, Maja K.; Oro, Anthony E.; Artandi, Steven E.

    2005-01-01

    TERT, the protein component of telomerase1,2, serves to maintain telomere function through the de novo addition of telomere repeats to chromosome ends and is reactivated in 90% of human cancers. In normal tissues, TERT is expressed in stem cells and in progenitor cells3, but its role in these compartments is not fully understood. Here, we show that conditional transgenic induction of TERT in mouse skin epithelium causes a rapid transition from telogen, the resting phase of the hair follicle cycle, to anagen, the active phase, thereby facilitating robust hair growth. TERT overexpression promotes this developmental transition by causing proliferation of quiescent, multipotent stem cells in the hair follicle bulge region. This new function for TERT does not require the telomerase RNA component (TERC), which encodes the template for telomere addition, and therefore operates through a novel mechanism independent of its activity in synthesizing telomere repeats. These data indicate that, in addition to its established role in extending telomeres, TERT can promote proliferation of resting stem cells through a non-canonical pathway. PMID:16107853

  20. Hoxb8 conditionally immortalised macrophage lines model inflammatory monocytic cells with important similarity to dendritic cells.

    PubMed

    Rosas, Marcela; Osorio, Fabiola; Robinson, Matthew J; Davies, Luke C; Dierkes, Nicola; Jones, Simon A; Reis e Sousa, Caetano; Taylor, Philip R

    2011-02-01

    We have examined the potential to generate bona fide macrophages (MØ) from conditionally immortalised murine bone marrow precursors. MØ can be derived from Hoxb8 conditionally immortalised macrophage precursor cell lines (MØP) using either M-CSF or GM-CSF. When differentiated in GM-CSF (GM-MØP) the resultant cells resemble GM-CSF bone marrow-derived dendritic cells (BMDC) in morphological phenotype, antigen phenotype and functional responses to microbial stimuli. In spite of this high similarity between the two cell types and the ability of GM-MØP to effectively present antigen to a T-cell hybridoma, these cells are comparatively poor at priming the expansion of IFN-γ responses from naïve CD4(+) T cells. The generation of MØP from transgenic or genetically aberrant mice provides an excellent opportunity to study the inflammatory role of GM-MØP, and reduces the need for mouse colonies in many studies. Hence differentiation of conditionally immortalised MØPs in GM-CSF represents a unique in vitro model of inflammatory monocyte-like cells, with important differences from bone marrow-derived dendritic cells, which will facilitate functional studies relating to the many 'sub-phenotypes' of inflammatory monocytes.

  1. Effect of metformin on Schwann cells under hypoxia condition

    PubMed Central

    Ma, Junxiong; Liu, Jun; Yu, Hailong; Chen, Yu; Wang, Qi; Xiang, Liangbi

    2015-01-01

    Metformin, which is the first-line drug for the treatment of diabetes mellitus type 2, has been proved to possess beneficial effects on nerve regeneration in many studies. However, the underlying mechanism is currently unclear. The present study was designed to investigate the potential beneficial effect of metformin on SCs under hypoxia condition, which is a biological process at the injury site. The cell number and cell viability of SCs were examined using fluorescence observation and MTT assay. The migration of SCs was evaluated using a Transwell chamber. The expression and secretion of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell derived neurotrophic factor (GDNF) and neural cell adhesion molecule (N-CAM) in SCs were assayed by RT-PCR and ELISA method. The results showed that metformin could help SCs recover from hypoxia injury and inhibit hypoxia-induced apoptosis. In addition, metformin could partially reverse the detrimental effect of hypoxia on cell number, viability, migration and adhesion. Metformin is also capable of maintaining the biological activities of SCs after hypoxia injury, such as increasing the expression and secretion of BDNF, NGF, GDNF, and N-CAM. Further studies showed that pre-incubation with AMPK (5’-AMP-activated protein kinase) inhibitor Compound C might partially inhibit the effect of metformin mentioned above, indicating the possible involvement of AMPK pathway in the beneficial effects of metformin on peripheral nervous system. In conclusion, metformin is capable of alleviating hypoxia-induced injury to SCs and AMPK pathway might be involved in this process. PMID:26261558

  2. Arteriolar niches maintain haematopoietic stem cell quiescence.

    PubMed

    Kunisaki, Yuya; Bruns, Ingmar; Scheiermann, Christoph; Ahmed, Jalal; Pinho, Sandra; Zhang, Dachuan; Mizoguchi, Toshihide; Wei, Qiaozhi; Lucas, Daniel; Ito, Keisuke; Mar, Jessica C; Bergman, Aviv; Frenette, Paul S

    2013-10-31

    Cell cycle quiescence is a critical feature contributing to haematopoietic stem cell (HSC) maintenance. Although various candidate stromal cells have been identified as potential HSC niches, the spatial localization of quiescent HSCs in the bone marrow remains unclear. Here, using a novel approach that combines whole-mount confocal immunofluorescence imaging techniques and computational modelling to analyse significant three-dimensional associations in the mouse bone marrow among vascular structures, stromal cells and HSCs, we show that quiescent HSCs associate specifically with small arterioles that are preferentially found in endosteal bone marrow. These arterioles are ensheathed exclusively by rare NG2 (also known as CSPG4)(+) pericytes, distinct from sinusoid-associated leptin receptor (LEPR)(+) cells. Pharmacological or genetic activation of the HSC cell cycle alters the distribution of HSCs from NG2(+) periarteriolar niches to LEPR(+) perisinusoidal niches. Conditional depletion of NG2(+) cells induces HSC cycling and reduces functional long-term repopulating HSCs in the bone marrow. These results thus indicate that arteriolar niches are indispensable for maintaining HSC quiescence.

  3. Hyperglycemic and Hyperlipidemic Conditions Alter Cardiac Cell Biomechanical Properties

    PubMed Central

    Michaelson, Jarett; Hariharan, Venkatesh; Huang, Hayden

    2014-01-01

    Currently, many diabetic cardiomyopathy (DC) studies focus on either in vitro molecular pathways or in vivo whole-heart properties such as ejection fraction. However, as DC is primarily a disease caused by changes in structural and functional properties, such studies may not precisely identify the influence of hyperglycemia or hyperlipidemia in producing specific cellular changes, such as increased myocardial stiffness or diastolic dysfunction. To address this need, we developed an in vitro approach to examine how structural and functional properties may change as a result of a diabetic environment. Particle-tracking microrheology was used to characterize the biomechanical properties of cardiac myocytes and fibroblasts under hyperglycemia or hyperlipidemic conditions. We showed that myocytes, but not fibroblasts, exhibited increased stiffness under diabetic conditions. Hyperlipidemia, but not hyperglycemia, led to increased cFos expression. Although direct application of reactive oxygen species had only limited effects that altered myocyte properties, the antioxidant N-acetylcysteine had broader effects in limiting glucose or fatty-acid alterations. Changes consistent with clinical DC alterations occur in cells cultured in elevated glucose or fatty acids. However, the individual roles of glucose, reactive oxygen species, and fatty acids are varied, suggesting multiple pathway involvement. PMID:24896111

  4. Hyperglycemic and hyperlipidemic conditions alter cardiac cell biomechanical properties.

    PubMed

    Michaelson, Jarett; Hariharan, Venkatesh; Huang, Hayden

    2014-06-03

    Currently, many diabetic cardiomyopathy (DC) studies focus on either in vitro molecular pathways or in vivo whole-heart properties such as ejection fraction. However, as DC is primarily a disease caused by changes in structural and functional properties, such studies may not precisely identify the influence of hyperglycemia or hyperlipidemia in producing specific cellular changes, such as increased myocardial stiffness or diastolic dysfunction. To address this need, we developed an in vitro approach to examine how structural and functional properties may change as a result of a diabetic environment. Particle-tracking microrheology was used to characterize the biomechanical properties of cardiac myocytes and fibroblasts under hyperglycemia or hyperlipidemic conditions. We showed that myocytes, but not fibroblasts, exhibited increased stiffness under diabetic conditions. Hyperlipidemia, but not hyperglycemia, led to increased cFos expression. Although direct application of reactive oxygen species had only limited effects that altered myocyte properties, the antioxidant N-acetylcysteine had broader effects in limiting glucose or fatty-acid alterations. Changes consistent with clinical DC alterations occur in cells cultured in elevated glucose or fatty acids. However, the individual roles of glucose, reactive oxygen species, and fatty acids are varied, suggesting multiple pathway involvement.

  5. A microfluidic live cell assay to study anthrax toxin induced cell lethality assisted by conditioned medium

    PubMed Central

    Shen, Jie; Cai, Changzu; Yu, Zhilong; Pang, Yuhong; Zhou, Ying; Qian, Lili; Wei, Wensheng; Huang, Yanyi

    2015-01-01

    It is technically challenging to investigate the function of secreted protein in real time by supply of conditioned medium that contains secreted protein of interest. The internalization of anthrax toxin is facilitated by a secreted protein Dickkopf-1 (DKK1) and its receptor, and eventually leads to cell lethality. To monitor the dynamic interplay between these components in live cells, we use an integrated microfluidic device to perform the cell viability assays with real-time controlled culture microenvironment in parallel. Conditioned medium, which contains the secreted proteins from specific cell lines, can be continuously pumped towards the cells that exposed to toxin. The exogenous DKK1 secreted from distant cells is able to rescue the sensitivity to toxin for those DKK1-knocked-down cells. This high-throughput assay allows us to precisely quantify the dynamic interaction between key components that cause cell death, and provide independent evidence of the function of DKK1 in the complex process of anthrax toxin internalization. PMID:25731605

  6. Toward Physiological Conditions for Cell Analyses: Forces of Heart Muscle Cells Suspended Between Elastic Micropillars☆

    PubMed Central

    Kajzar, A.; Cesa, C.M.; Kirchgeßner, N.; Hoffmann, B.; Merkel, R.

    2008-01-01

    Almost each mammalian cell permanently applies forces to its environment. These forces are essential for many vital processes such as tissue formation or cell movement. In turn, the environmental conditions of cells strongly affect force production. Here we report on the development of an array of elastomeric micropillars as cellular environment. Within these micropillar arrays, we cultivated rat heart muscle cells (cardiac myocytes). For lattice constants between 20 and 30 μm, cells strongly preferred spanning between the elastic micropillars over adhering to the underlying flat substrate. In addition, the architectures of the cytoskeleton and of protein complexes formed for adhesion were strongly dependent on the environment of the cell. On flat parts of the substrates, we observed prominent stress fibers and focal adhesion sites. In contrast, cells suspended between micropillars exhibited well organized myofibers and costameric adhesions at the locations of Z-bands. These observations argue for close-to-nature environmental conditions within micropillar arrays. Resting as well as contraction forces of myocytes resulted in measurable pillar bending. Using an approximate theoretical treatment of elastically founded micropillars, we calculated average cell forces of 140 nN in the relaxed and 400 nN in the contracted state. PMID:17981895

  7. Inhibiting stromal cell heparan sulfate synthesis improves stem cell mobilization and enables engraftment without cytotoxic conditioning

    PubMed Central

    Saez, Borja; Ferraro, Francesca; Yusuf, Rushdia Z.; Cook, Colleen M.; Yu, Vionnie W. C.; Pardo-Saganta, Ana; Sykes, Stephen M.; Palchaudhuri, Rahul; Schajnovitz, Amir; Lotinun, Sutada; Lymperi, Stefania; Mendez-Ferrer, Simon; del Toro, Raquel; Day, Robyn; Vasic, Radovan; Acharya, Sanket S.; Baron, Roland; Lin, Charles P.; Yamaguchi, Yu; Wagers, Amy J.

    2014-01-01

    The glycosyltransferase gene, Ext1, is essential for heparan sulfate production. Induced deletion of Ext1 selectively in Mx1-expressing bone marrow (BM) stromal cells, a known population of skeletal stem/progenitor cells, in adult mice resulted in marked changes in hematopoietic stem and progenitor cell (HSPC) localization. HSPC egressed from BM to spleen after Ext1 deletion. This was associated with altered signaling in the stromal cells and with reduced vascular cell adhesion molecule 1 production by them. Further, pharmacologic inhibition of heparan sulfate mobilized qualitatively more potent and quantitatively more HSPC from the BM than granulocyte colony-stimulating factor alone, including in a setting of granulocyte colony-stimulating factor resistance. The reduced presence of endogenous HSPC after Ext1 deletion was associated with engraftment of transfused HSPC without any toxic conditioning of the host. Therefore, inhibiting heparan sulfate production may provide a means for avoiding the toxicities of radiation or chemotherapy in HSPC transplantation for nonmalignant conditions. PMID:25202142

  8. Germ-cell culture conditions facilitate the production of mouse embryonic stem cells.

    PubMed

    Ramos-Ibeas, Priscila; Pericuesta, Eva; Fernández-González, Raúl; Gutiérrez-Adán, Alfonso; Ramírez, Miguel Ángel

    2014-09-01

    The derivation of embryonic stem-cell (ESC) lines from blastocysts is a very inefficient process. Murine ESCs are thought to arise from epiblast cells that are already predisposed to a primordial-germ-cell fate. During the process of ESC derivation from B6D2 F1 hybrid mice, if we first culture the embryo from the two-cell stage in medium supplemented with LIF, we improve the quality of the blastocyst. When the blastocyst is then cultured in a germ-line stem-cell culture medium (GSCm), we are able to more efficiently (28.3%) obtain quality ESC lines that have a normal karyotype, proper degree of chimerism, and exhibit germ-line transmission when microinjected into blastocysts. Although germ-cell-specific genes were expressed in all culture medium conditions, GSCm did not shift the transcriptome towards germ-cell specification. A correlation was further observed between ESC derivation efficiency and the expression of some imprinted genes and retrotransposable elements. In conclusion, the combination of LIF supplementation followed by culture in GSCm establishes a higher efficiency method for ESC derivation.

  9. Enhancing the Efficacy of Dendritic Cell Vaccines by Tissue Conditioning

    DTIC Science & Technology

    2009-03-01

    Kubler , H., Tseng, T., Sakai, Y., Su, Z. et al.: Oxidative Stress Regulates Expression of VEGFR1 in Myeloid Cells: Link to Tumor-Induced Immune Suppression in Renal Cell Carcinoma. J Immunol, 181: 346, 2008

  10. Propagation of Human Embryonic Stem Cells on Human Amniotic Fluid Cells as Feeder Cells in Xeno-Free Culture Conditions

    PubMed Central

    Jung, Juwon; Baek, Jin Ah; Seol, Hye Won; Choi, Young Min

    2016-01-01

    Human embryonic stem cells (hESCs) have been routinely cultured on mouse embryonic fibroblast feederlayers with a medium containing animal materials. For clinical application of hESCs, animal-derived products from the animal feeder cells, animal substrates such as gelatin or Matrigel and animal serum are strictly to be eliminated in the culture system. In this study, we performed that SNUhES32 and H1 were cultured on human amniotic fluid cells (hAFCs) with KOSR XenoFree and a humanized substrate. All of hESCs were relatively well propagated on hAFCs feeders with xeno-free conditions and they expressed pluripotent stem cell markers, alkaline phosphatase, SSEA-4, TRA1-60, TRA1-81, Oct-4, and Nanog like hESCs cultured on STO or human foreskin fibroblast feeders. In addition, we observed the expression of nonhuman N-glycolylneuraminic acid (Neu5GC) molecules by flow cytometry, which was xenotransplantation components of contamination in hESCs cultured on animal feeder conditions, was not detected in this xeno-free condition. In conclusion, SNUhES32 and H1 could be maintained on hAFCs for humanized culture conditions, therefore, we suggested that new xenofree conditions for clinical grade hESCs culture will be useful data in future clinical studies. PMID:27294211

  11. Conditions of steady switching in phase-transition memory cells

    SciTech Connect

    Popov, A. I. Salnikov, S. M.; Anufriev, Yu. V.

    2015-04-15

    Three types of non-volatile memory cells of different designs based on phase transitions are developed and implemented. The effect of the design features of the cells and their active-region sizes on the switching characteristics and normal operation of the cells is considered as a whole. The causes of failure of the cells are analyzed from the obtained series of scanning electron images upon level-by-level etching of the samples. It is shown that the cell design is the most critical factor from the viewpoint of switching to the high-resistance state. The causes of this fact are analyzed and the criterion for providing the steady operation of cells of non-volatile memory based on phase transitions is formulated.

  12. Porcine tooth germ cell conditioned medium can induce odontogenic differentiation of human dental pulp stem cells.

    PubMed

    Wang, Yin-Xiong; Ma, Zhao-Feng; Huo, Na; Tang, Liang; Han, Chun; Duan, Yin-Zhong; Jin, Yan

    2011-05-01

    It is suggested that the differentiation of tooth-derived stem cells is modulated by the local microenvironment in which they reside. Previous studies have indicated that tooth germ cell-conditioned medium (TGC-CM) holds the potential to induce dental pulp stem cells (DPSCs) to differentiate into the odontogenic lineage. Nevertheless, human TGC-CM (hTGC-CM) is not feasible in practical application, so we conjectured that xenogenic TGC-CM might exert a similar influence on human dental stem cells. In this study, we chose swine as the xenogenic origin and compared the effect of porcine tooth germ cell-conditioned medium (pTGC-CM) with its human counterpart on human DPSCs. Morphological appearance, colony-forming assay, in vitro multipotential ability, protein and gene expression of the odontogenic phenotype and the in vivo differentiation capacity of DPSCs were evaluated. The results showed that pTGC-CM exerted a similar effect to hTGC-CM in inducing human DPSCs to present odontogenic changes, which were indicated by remarkable morphological changes, higher multipotential capability and the expression of some odontogenic markers in gene and protein levels. Besides, the in vivo results showed that pTGC-CM-treated DPSCs, similar to hTGC-CM-treated DPSCs, could form a more regular dentine-pulp complex. Our data provided the first evidence that pTGC-CM is able to exert almost the same effect on DPSCs with hTGC-CM. The observations suggest that the application of xenogenic TGC-CM may facilitate generating bioengineered teeth from tooth-derived stem cells in future.

  13. Sonoporation of adherent cells under regulated ultrasound cavitation conditions.

    PubMed

    Muleki Seya, Pauline; Fouqueray, Manuela; Ngo, Jacqueline; Poizat, Adrien; Inserra, Claude; Béra, Jean-Christophe

    2015-04-01

    A sonoporation device dedicated to the adherent cell monolayer has been implemented with a regulation process allowing the real-time monitoring and control of inertial cavitation activity. Use of the cavitation-regulated device revealed first that adherent cell sonoporation efficiency is related to inertial cavitation activity, without inducing additional cell mortality. Reproducibility is enhanced for the highest sonoporation rates (up to 17%); sonoporation efficiency can reach 26% when advantage is taken of the standing wave acoustic configuration by applying a frequency sweep with ultrasound frequency tuned to the modal acoustic modes of the cavity. This device allows sonoporation of adherent and suspended cells, and the use of regulation allows some environmental parameters such as the temperature of the medium to be overcome, resulting in the possibility of cell sonoporation even at ambient temperature.

  14. Characterization of Glial Cell Models and In Vitro Manipulation of the Neuregulin1/ErbB System

    PubMed Central

    2014-01-01

    The neuregulin1/ErbB system plays an important role in Schwann cell behavior both in normal and pathological conditions. Upon investigation of the expression of the neuregulin1/ErbB system in vitro, we explored the possibility to manipulate the system in order to increase the migration of Schwann cells, that play a fundamental role in the peripheral nerve regeneration. Comparison of primary cells and stable cell lines shows that both primary olfactory bulb ensheathing cells and a corresponding cell line express ErbB1-ErbB2 and neuregulin1, and that both primary Schwann cells and a corresponding cell line express ErbB2-ErbB3, while only primary Schwann cells express neuregulin1. To interfere with the neuregulin1/ErbB system, the soluble extracellular domain of the neuregulin1 receptor ErbB4 (ecto-ErbB4) was expressed in vitro in the neuregulin1 expressing cell line, and an unexpected increase in cell motility was observed. In vitro experiments suggest that the back signaling mediated by the transmembrane neuregulin1 plays a role in the migratory activity induced by ecto-ErbB4. These results indicate that ecto-ErbB4 could be used in vivo as a tool to manipulate the neuregulin1/ErbB system. PMID:25177687

  15. Characterization of glial cell models and in vitro manipulation of the neuregulin1/ErbB system.

    PubMed

    Pascal, Davide; Giovannelli, Alessia; Gnavi, Sara; Hoyng, Stefan Adriaan; de Winter, Fred; Morano, Michela; Fregnan, Federica; Dell'Albani, Paola; Zaccheo, Damiano; Perroteau, Isabelle; Pellitteri, Rosalia; Gambarotta, Giovanna

    2014-01-01

    The neuregulin1/ErbB system plays an important role in Schwann cell behavior both in normal and pathological conditions. Upon investigation of the expression of the neuregulin1/ErbB system in vitro, we explored the possibility to manipulate the system in order to increase the migration of Schwann cells, that play a fundamental role in the peripheral nerve regeneration. Comparison of primary cells and stable cell lines shows that both primary olfactory bulb ensheathing cells and a corresponding cell line express ErbB1-ErbB2 and neuregulin1, and that both primary Schwann cells and a corresponding cell line express ErbB2-ErbB3, while only primary Schwann cells express neuregulin1. To interfere with the neuregulin1/ErbB system, the soluble extracellular domain of the neuregulin1 receptor ErbB4 (ecto-ErbB4) was expressed in vitro in the neuregulin1 expressing cell line, and an unexpected increase in cell motility was observed. In vitro experiments suggest that the back signaling mediated by the transmembrane neuregulin1 plays a role in the migratory activity induced by ecto-ErbB4. These results indicate that ecto-ErbB4 could be used in vivo as a tool to manipulate the neuregulin1/ErbB system.

  16. Production of Plasminogen Activator in Cultures of Superior Cervical Ganglia and Isolated Schwann Cells

    NASA Astrophysics Data System (ADS)

    Alvarez-Buylla, Arturo; Valinsky, Jay E.

    1985-05-01

    Plasminogen activator has been implicated in tissue remodeling and cell migration during embryogenesis. In the developing nervous system, these processes are evident in the migration of neurons, axonal extension, Schwann cell migration, and the ensheathment and myelination of nerves. We have studied the production of plasminogen activator in cultures of superior cervical ganglia under conditions in which both neurons and glia are present. We have found that a principal source of the enzyme in these cultures is the glial cells and that the enzyme could not be detected at the growing tips of neurites. Plasminogen activator is also produced by Schwann cells isolated from neonatal rat sciatic nerve. The production of the enzyme by these cells is stimulated 6- to 10-fold by cholera toxin. Isolated Schwann cells and glial cells in the ganglion explant cultures produce the tissue form of plasminogen activator, a form of the enzyme not often found in nonmalignant cells. Preliminary experiments suggest that neuronal-glial interactions may regulate enzyme production by Schwann cells.

  17. Stem cell transplantation after reduced-intensity conditioning for sickle cell disease.

    PubMed

    Matthes-Martin, Susanne; Lawitschka, Anita; Fritsch, Gerhard; Lion, Thomas; Grimm, Brigitte; Breuer, Sabine; Boztug, Heidrun; Karlhuber, Susanne; Holter, Wolfgang; Peters, Christina; Minkov, Milen

    2013-04-01

    Sickle cell disease (SCD) is still associated with substantial morbidity and reduced life expectancy. Disease-related mortality rises to 14% in adolescents and young adults. Overall and disease-free survival following haematopoietic stem cell transplantation (HSCT) is 90% and 95%, respectively. To reduce transplant-associated late effects, the feasibility of a highly immunosuppressive reduced-intensity conditioning (RIC) regimen was explored in children with SCD and a matched sibling donor. Eight patients (median age, 9 yr) and symptomatic SCD were included. The conditioning regimen consisted of fludarabine, melphalan and either thiotepa or total lymphoid irradiation plus antithymocyte globuline or alemtuzumab. The graft was bone marrow in seven and cord blood in one case. The conditioning regimen was well tolerated and no severe infectious complications occurred. All patients displayed mixed chimaerism on day +28. After a median follow-up of 4 yr, 3/8 patients have mixed leucocyte chimaerism and 8/8 patients have 100% donor erythropoiesis. HSCT from matched sibling donors following a RIC regimen was well tolerated and resulted in cure in all patients studied. If confirmed in larger patient cohorts, these observations will have important implications for the indications of HSCT in children with SCD.

  18. Defining cell culture conditions to improve human norovirus infectivity assays.

    PubMed

    Straub, T M; Hutchison, J R; Bartholomew, R A; Valdez, C O; Valentine, N B; Dohnalkova, A; Ozanich, R M; Bruckner-Lea, C J

    2013-01-01

    Significant difficulties remain for determining whether human noroviruses (hNoV) recovered from water, food, and environmental samples are infectious. Three-dimensional (3-D) tissue culture of human intestinal cells has shown promise in developing an infectivity assay, but reproducibility, even within a single laboratory, remains problematic. From the literature and our observations, we hypothesized that the common factors that lead to more reproducible hNoV infectivity in vitro requires that the cell line be (1) of human gastrointestinal origin, (2) expresses apical microvilli, and (3) be a positive secretor cell line. The C2BBe1 cell line, which is a brush-border producing clone of Caco-2, meets these three criteria. When challenged with Genogroup II viruses, we observed a 2 Log(10) increase in viral RNA titer. A passage experiment with GII viruses showed evidence of the ability to propagate hNoV by both quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microscopy. In our hands, using 3-D C2BBe1 cells improves reproducibility of the infectivity assay for hNoV, but the assay can still be variable. Two sources of variability include the cells themselves (mixed phenotypes of small and large intestine) and initial titer measurements using qRT-PCR that measures all RNA vs. plaque assays that measure infectious virus.

  19. Defining cell culture conditions to improve human norovirus infectivity assays

    SciTech Connect

    Straub, Tim M.; Hutchison, Janine R.; Bartholomew, Rachel A.; Valdez, Catherine O.; Valentine, Nancy B.; Dohnalkova, Alice; Ozanich, Richard M.; Bruckner-Lea, Cindy J.

    2013-01-10

    Significant difficulties remain for determining whether human noroviruses (hNoV) recovered from water, food, and environmental samples are infectious. Three-dimensional tissue culture of human intestinal cells has shown promise in developing an infectivity assay, but reproducibility, even within a single laboratory, remains problematic. From the literature and our observations, we hypothesized that the common factors that leads to more reproducible hNoV infectivity in vitro requires that the cell line be 1) of human gastrointestinal origin, 2) expresses apical microvilli, and 3) be a positive secretor cell line. The C2BBe1 cell line, which is a brush-border producing clone of Caco-2, meets these three criteria. When challenged with Genogroup II viruses, we observed a 2 Log10 increase in viral RNA titer. A passage experiment with GII viruses showed evidence of the ability to propagate hNoV by both reverse transcription quantitative PCR (qRT-PCR) and microscopy. Using 3-D C2BBe1 cells improves reproducibility of the infectivity assay for hNoV, but the assay can still be variable. Two sources of variability include the cells themselves (mixed phenotypes of small and large intestine) and initial titer measurements using quantitative reverse transcription PCR (qRT-PCR) that measures all RNA vs. plaque assays that measure infectious virus.

  20. Encapsulation of Perovskite Solar Cells for High Humidity Conditions.

    PubMed

    Dong, Qi; Liu, Fangzhou; Wong, Man Kwong; Tam, Ho Won; Djurišić, Aleksandra B; Ng, Annie; Surya, Charles; Chan, Wai Kin; Ng, Alan Man Ching

    2016-09-22

    We examined different encapsulation strategies for perovskite solar cells by testing the device stability under continuous illumination, elevated temperature (85 °C) and ambient humidity of 65 %. The effects of the use of different epoxies, protective layers and the presence of desiccant were investigated. The best stability (retention of ∼80 % of initial efficiency on average after 48 h) was obtained for devices protected by a SiO2 film and encapsulated with a UV-curable epoxy including a desiccant sheet. However, the stability of ZnO-based cells encapsulated by the same method was found to be inferior to that of TiO2 -based cells. Finally, outdoor performance tests were performed for TiO2 -based cells (30-90 % ambient humidity). All the stability tests were performed following the established international summit on organic photovoltaic stability (ISOS) protocols for organic solar cell testing (ISOS-L2 and ISOS-O1).

  1. Can manipulation of differentiation conditions eliminate proliferative cells from a population of ES cell-derived forebrain cells?

    PubMed Central

    Precious, Sophie V.; Kelly, Claire M.; Allen, Nicholas D.; Rosser, Anne E.

    2016-01-01

    ABSTRACT There is preliminary evidence that implantation of primary fetal striatal cells provides functional benefit in patients with Huntington's disease, a neurodegenerative condition resulting in loss of medium-sized spiny neurons (MSN) of the striatum. Scarcity of primary fetal tissue means it is important to identify a renewable source of cells from which to derive donor MSNs. Embryonic stem (ES) cells, which predominantly default to telencephalic-like precursors in chemically defined medium (CDM), offer a potentially inexhaustible supply of cells capable of generating the desired neurons. Using an ES cell line, with the forebrain marker FoxG1 tagged to the LacZ reporter, we assessed effects of known developmental factors on the yield of forebrain-like precursor cells in CDM suspension culture. Addition of FGF2, but not DKK1, increased the proportion of FoxG1-expressing cells at day 8 of neural induction. Oct4 was expressed at day 8, but was undetectable by day 16. Differentiation of day 16 precursors generated GABA-expressing neurons, with few DARPP32 positive MSNs. Transplantation of day 8 precursor cells into quinolinic acid-lesioned striata resulted in generation of teratomas. However, transplantation of day 16 precursors yielded grafts expressing neuronal markers including NeuN, calbindin and parvalbumin, but no DARPP32 6 weeks post-transplantation. Manipulation of fate of ES cells requires optimization of both concentration and timing of addition of factors to culture systems to generate the desired phenotypes. Furthermore, we highlight the value of increasing the precursor phase of ES cell suspension culture when directing differentiation toward forebrain fate, so as to dramatically reduce the risk of teratoma formation. PMID:27606335

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  3. Effective donor cell fusion conditions for production of cloned dogs by somatic cell nuclear transfer.

    PubMed

    Park, JungEun; Oh, HyunJu; Hong, SoGun; Kim, MinJung; Kim, GeonA; Koo, OkJae; Kang, SungKeun; Jang, Goo; Lee, ByeongChun

    2011-03-01

    As shown by the birth of the first cloned dog 'Snuppy', a protocol to produce viable cloned dogs has been reported. In order to evaluate optimum fusion conditions for improving dog cloning efficiency, in vivo matured oocytes were reconstructed with adult somatic cells from a female Pekingese using different fusion conditions. Fusion with needle vs chamber methods, and with low vs high pulse strength was compared by evaluating fusion rate and in vivo development of canine cloned embryos. The fusion rates in the high voltage groups were significantly higher than in the low voltage groups regardless of fusion method (83.5 vs 66.1% for the needle fusion method, 67.4 vs 37.9% for the fusion chamber method). After embryo transfer, one each pregnancy was detected after using the needle fusion method with high and low voltage and in the chamber fusion method with high voltage, whereas no pregnancy was detected using the chamber method with low voltage. However, only the pregnancy from the needle fusion method with high voltage was maintained to term and one healthy puppy was delivered. The results of the present study demonstrated that two DC pulses of 3.8 to 4.0 kV/cm for 15 μsec using the needle fusion method were the most effective method for the production of cloned dogs under the conditions of this experiment.

  4. Influence of Mesenchymal Stem Cells Conditioned Media on Proliferation of Urinary Tract Cancer Cell Lines and Their Sensitivity to Ciprofloxacin.

    PubMed

    Maj, Malgorzata; Bajek, Anna; Nalejska, Ewelina; Porowinska, Dorota; Kloskowski, Tomasz; Gackowska, Lidia; Drewa, Tomasz

    2017-06-01

    Mesenchymal stem cells (MSCs) are known to interact with cancer cells through direct cell-to-cell contact and secretion of paracrine factors, although their exact influence on tumor progression in vivo remains unclear. To better understand how fetal and adult stem cells affect tumors, we analyzed viability of human renal (786-0) and bladder (T24) carcinoma cell lines cultured in conditioned media harvested from amniotic fluid-derived stem cells (AFSCs) and adipose-derived stem cells (ASCs). Both media reduced metabolic activity of 786-0 cells, however, decreased viability of T24 cells was noted only after incubation with conditioned medium from ASCs. To test the hypothesis that MSCs-secreted factors might be involved in chemoresistance acquisition, we further analyzed influence of mesenchymal stem cell conditioned media (MSC-CM) on cancer cells sensitivity to ciprofloxacin, that is considered as potential candidate agent for urinary tract cancers treatment. Significantly increased resistance to tested drug indicates that MSCs may protect cancer cells from chemotherapy. J. Cell. Biochem. 118: 1361-1368, 2017. © 2016 Wiley Periodicals, Inc.

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

    PubMed

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

    2013-10-24

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

  6. Nonlinear Dielectric Properties of Yeast Cells Cultured in Different Environmental Conditions

    NASA Astrophysics Data System (ADS)

    Kawanishi, Gomon; Fukuda, Naoki; Muraji, Masafumi

    The harmonics of the electric current through yeast suspensions, the nonlinear dielectric properties of yeast cells, have particular patterns according to the biological activity of the cells and the measurement of these patterns is a technique for determining the activity of living cells. The concentration of glucose and oxygen in yeast culture medium influences the manifestation of fermentation or respiration of yeast cells. Measurements were made with yeast cells (Saccharomyces cerevisiae) cultured aerobically and anaerobically in sufficient glucose concentration, aerobic fermentation and anaerobic fermentation, and aerobically in limited glucose concentration, respiration. The results showed that the harmonics were barely apparent for yeast cells in aerobic fermentation and respiratory; however, cells in the anaerobic fermentation displayed substantial third and fifth harmonics. We can say that environmental condition affects the yeast cells' nonlinear properties, from another viewpoint, the measurements of the nonlinear properties are available to determine the activity of yeast cells adjusted to the conditions of their cultivation.

  7. Parameter identification for model of T cell proliferation in lymphopenia conditions.

    PubMed

    Ayoub, H; Ainseba, B E; Langlais, M; Hogan, T; Callard, R; Seddon, B; Thiébaut, R

    2014-05-01

    The number of T Lymphocytes (T cells) in the body is under homeostatic control. At equilibrium, the majority of naive T cells are non-dividing and express low levels of the surface protein CD44. In conditions of T cell deficiency (lymphopenia), naive T cells enter into a proliferative phase, undergoing cell division accompanied by a subtle change in their surface expression of CD44. In this study, we use a mathematical modelling approach to analyse the proliferative response of transgenic T cells in lymphopenic conditions. Our nonlinear model is composed of ordinary differential equations and partial differential equations structured by age (maturity of cell) and CD44 expression. To better understand the evolution of CD44 expression on the surface of T cells during cell division, we present a numerical analysis to solve a parameter identification problem. Finally, we show the parameters and the simulations that we obtain from the model and compare them to experimental data.

  8. Renal differentiation of Mesenchymal stem cells seeded on nanofibrous scaffolds improved by Human renal tubular cell lines conditioned medium.

    PubMed

    Ardeshirylajimi, Abdolreza; Vakilian, Saeid; Salehi, Mohammad

    2016-11-09

    Kidney injuries and renal dysfunctions are one of the most important clinical problems and tissue engineering could be a valuable method for solving it. The objective of this study was to investigate the synergistic effect of renal cell line conditioned medium and Polycaprolactone nanofibers on renal differentiation of human mesenchymal stem cells. In the present study, after stem cells isolation and characterization, Polycaprolactone nanofibrous scaffold was fabricated using electrospinning methods and characterized morphologically, mechanically and biocompatibility. And then the renal differentiation of seeded mesenchymal stem cells on the surface of Polycaprolactone nanofibers with and without human renal tubular cell lines conditioned medium was investigated by evaluation of eight important renal related genes expression by Real-time RT-PCR and immunocytochemistry. Fabricated nanofibrous scaffolds were good in all characterized items. Almost highest expression of all genes was detected in stem cells seeded on Polycaprolactone under conditioned media in comparison with the stem cells seeded on Polycaprolactone, tissue culture polystyrene under renal induction medium and tissue culture polystyrene under conditioned medium. According to the results, Polycaprolactone nanofibers in contribution with conditioned medium can provide the optimal conditions for renal differentiation of mesenchymal stem cells and could be a promising candidate for renal tissue engineering application.

  9. Stroma-conditioned media improve expansion of human primitive hematopoietic stem cells and progenitor cells.

    PubMed

    Breems, D A; Blokland, E A; Ploemacher, R E

    1997-01-01

    It has been reported that stroma-dependent cultures support proliferation of hematopoietic stem cells (HSC). In order to investigate the effect of soluble stromal factors, we developed short-term serum-low liquid cultures in which the effect of stroma-conditioned media (SCM) from the murine FBMD-1, and human L87/4 and L88/5 cell lines was studied on the maintenance and expansion of various human HSC subsets in CD34-positive selected mobilized peripheral blood stem cells (PBSC) from autologous transplants of lymphoma and multiple myeloma patients. The human cobblestone area forming cell (CAFC) assay was employed to determine the frequencies of both the CAFC weeks 2 to 4 as tentative indicators of progenitor and transiently repopulating HSC, and the more primitive CAFC weeks 6 to 8 as indicators of long-term repopulating HSC. In 7-day liquid cultures containing interleukin-3 (IL-3), stem cell factor (SCF) and IL-6, we recovered 3.0-fold more colony-forming cells (CFC) and 1.7- to 1.9-fold more CAFC weeks 2 and 4. The absolute number of primitive CAFC weeks 6 and 8 were only maintained (1.1- to 1.4-fold) in these liquid cultures. This modest expansion was significantly improved by the addition of SCM from the FBMD-1, L87/4 or L88/5 cell lines. Output CFC numbers were 6.8-, 5.8- and 9.9-fold higher, respectively, than the input values, while absolute CAFC week 2 to 4 numbers were 4.5-, 10.2- and 10.2-fold expanded, respectively. The addition of SCM also improved expansion of the more primitive CAFC week 6 to 8 stem cell subsets by 2.2-, 4.5- and 4.9-fold, respectively. The addition of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF), IL-1beta, IL-11 or macrophage inflammatory protein-1alpha to cultures containing IL-3, SCF and IL-6 could not explain the SCM effect and in all these combinations SCM addition further increased the recovery of HSC subsets. Similarly, addition of anti-cytokine antibodies (ie alpha-G-CSF, alpha-GM-CSF, alpha

  10. Hydrogen generation at ambient conditions: application in fuel cells.

    PubMed

    Boddien, Albert; Loges, Björn; Junge, Henrik; Beller, Matthias

    2008-01-01

    The efficient generation of hydrogen from formic acid/amine adducts at ambient temperature is demonstrated. The highest catalytic activity (TOF up to 3630 h(-1) after 20 min) was observed in the presence of in situ generated ruthenium phosphine catalysts. Compared to the previously known methods to generate hydrogen from liquid feedstocks, the systems presented here can be operated at room temperature without the need for any high-temperature reforming processes, and the hydrogen produced can then be directly used in fuel cells. A variety of Ru precursors and phosphine ligands were investigated for the decomposition of formic acid/amine adducts. These catalytic systems are particularly interesting for the generation of H2 for new applications in portable electric devices.

  11. Retinoic acid combined with spermatogonial stem cell conditions facilitate the generation of mouse germ-like cells.

    PubMed

    Dong, Guoyi; Shang, Zhouchun; Liu, Longqi; Liu, Chuanyu; Ge, Yuping; Wang, Quanlei; Wu, Liang; Chen, Fang; Li, Baolin; Liu, Xin; Xu, Xun; Yang, Huanming; DU, Yutao; Jiang, Hui

    2017-03-17

    Spermatogenic lineage has been directly generated in spermatogonial stem cell (SSC) conditions from human pluripotent stem cells (PSCs). However, it remains unknown whether mouse ESCs can directly differentiate into advanced male germ cell lineage in the same conditions. Here, we showed that rather low efficiency of germ-like cell generation from mouse ESCs in SSC conditions. Interestingly, addition of retinoic acid (RA) into SSC conditions enabled efficient differentiation of mouse ESCs into germ-like cells, as shown by activating the expression of spermatogenesis-associated genes during differentiation process, such as Mvh , Dazl , Prdm14 , Stella , Scp1 , Scp3 , Stra8 and Rec8 In contrast, for cells cultured in control medium, the activation of these above genes barely occurred. In addition, RA with SSC conditions yielded colonies of Acrosin expressing cells and the positive ratio reached a peak at day 6. Our work thus establishes a simple and cost efficient approach for male germ-like cell differentiation from mouse pluripotent stem cells and may propose a useful strategy for studying spermatogenesis in vitro.

  12. Selective advantage of trisomic human cells cultured in non-standard conditions

    PubMed Central

    Rutledge, Samuel D.; Douglas, Temple A.; Nicholson, Joshua M.; Vila-Casadesús, Maria; Kantzler, Courtney L.; Wangsa, Darawalee; Barroso-Vilares, Monika; Kale, Shiv D.; Logarinho, Elsa; Cimini, Daniela

    2016-01-01

    An abnormal chromosome number, a condition known as aneuploidy, is a ubiquitous feature of cancer cells. A number of studies have shown that aneuploidy impairs cellular fitness. However, there is also evidence that aneuploidy can arise in response to specific challenges and can confer a selective advantage under certain environmental stresses. Cancer cells are likely exposed to a number of challenging conditions arising within the tumor microenvironment. To investigate whether aneuploidy may confer a selective advantage to cancer cells, we employed a controlled experimental system. We used the diploid, colorectal cancer cell line DLD1 and two DLD1-derived cell lines carrying single-chromosome aneuploidies to assess a number of cancer cell properties. Such properties, which included rates of proliferation and apoptosis, anchorage-independent growth, and invasiveness, were assessed both under standard culture conditions and under conditions of stress (i.e., serum starvation, drug treatment, hypoxia). Similar experiments were performed in diploid vs. aneuploid non-transformed human primary cells. Overall, our data show that aneuploidy can confer selective advantage to human cells cultured under non-standard conditions. These findings indicate that aneuploidy can increase the adaptability of cells, even those, such as cancer cells, that are already characterized by increased proliferative capacity and aggressive tumorigenic phenotypes. PMID:26956415

  13. Model-based condition monitoring of PEM fuel cell using Hotelling T 2 control limit

    NASA Astrophysics Data System (ADS)

    Xue, X.; Tang, J.; Sammes, N.; Ding, Y.

    Although a variety of design and control strategies have been proposed to improve the performance of polymer electrolyte membrane (PEM) fuel cell systems, temporary faults in such systems still might occur during operations due to the complexity of the physical process and the functional limitations of some components. The development of an effective condition monitoring system that can detect these faults in a timely manner is complicated by the operating condition variation, the significant variability/uncertainty of the fuel cell system, and the measurement noise. In this research, we propose a model-based condition monitoring scheme that employs the Hotelling T 2 statistical analysis for fault detection of PEM fuel cells. Under a given operating condition, the instantaneous load current, the temperature and fuel/gas source pressures of the fuel cell are measured. These measurements are then fed into a lumped parameter dynamic fuel cell model for the establishment of the baseline under the same operating condition for comparison. The fuel cell operation is simulated under statistical sampling of parametric uncertainties with specified statistics (mean and variance) that account for the system variability/uncertainty and measurement noise. This yields a group of output voltages (under the same operating condition but with uncertainties) as the baseline. Fault detection is facilitated by comparing the real-time measurement of the fuel cell output voltage with the baseline voltages by employing the Hotelling T 2 statistical analysis. The baseline voltages are used to evaluate the output T 2 statistics under normal operating condition. Then, with a given confidence level the upper control limit can be specified. Fault condition will be declared if the T 2 statistics of real-time voltage measurement exceeds the upper control limit. This model-based robust condition monitoring scheme can deal with the operating condition variation, various uncertainties in a fuel cell

  14. Schwann cell migration and neurite outgrowth are influenced by media conditioned by epineurial fibroblasts.

    PubMed

    van Neerven, S G A; Pannaye, P; Bozkurt, A; Van Nieuwenhoven, F; Joosten, E; Hermans, E; Taccola, G; Deumens, R

    2013-11-12

    The regenerative capacity of the peripheral nervous system is largely related to Schwann cells undergoing proliferation and migration after injury and forming growth-supporting substrates for severed axons. Novel data show that fibroblasts to a certain extent regulate the pro-regenerative behavior of Schwann cells. In the setting of peripheral nerve injury, the fibroblasts that form the epineurium come into close contact with both Schwann cells and peripheral axons, but the potential influence on these latter two cell types has not been studied yet. In the present study we explored whether culture media, conditioned by epineurial fibroblasts can influence Schwann cells and/or neurite outgrowth from dorsal root ganglia neurons in vitro. Our data indicate that epineurial fibroblast-conditioned culture media substantially increase Schwann cell migration and the outgrowth of neurites. Schwann cell proliferation remained largely unaffected. These same read-out parameters were assayed in a condition where epineurial fibroblasts were subjected to stretch-cell-stress, a mechanical stressor that plays an important role in traumatic peripheral nerve injuries. Stretch-cell-stress of epineurial fibroblasts did not further change the positive effects of conditioned media on Schwann cell migration and neurite outgrowth. From these data we conclude that an as yet unknown pro-regenerative role can be attributed to epineurial fibroblasts, implying that such cells may affect the outcome of severe peripheral nerve injury.

  15. Single cell stiffness measurement at various humidity conditions by nanomanipulation of a nano-needle.

    PubMed

    Shen, Yajing; Nakajima, Masahiro; Yang, Zhan; Tajima, Hirotaka; Najdovski, Zoran; Homma, Michio; Fukuda, Toshio

    2013-04-12

    This paper presents a method for single cell stiffness measurement based on a nano-needle and nanomanipulation. The nano-needle with a buffering beam was fabricated from an atomic force microscope cantilever by the focused ion beam etching technique. Wild type yeast cells (W303) were prepared and placed on the sample stage inside an environmental scanning electron microscope (ESEM) chamber. The nanomanipulator actuated the nano-needle to press against a single yeast cell. As a result, the deformation of the cell and nano-needle was observed by the ESEM system in real-time. Finally, the stiffness of the single cell was determined based on this deformation information. To reveal the relationship between the cell stiffness and the environmental humidity conditions, the cell stiffness was measured at three different humidity conditions, i.e. 40, 70 and 100%, respectively. The results show that the stiffness of a single cell is reduced with increasing humidity.

  16. Single cell stiffness measurement at various humidity conditions by nanomanipulation of a nano-needle

    NASA Astrophysics Data System (ADS)

    Shen, Yajing; Nakajima, Masahiro; Yang, Zhan; Tajima, Hirotaka; Najdovski, Zoran; Homma, Michio; Fukuda, Toshio

    2013-04-01

    This paper presents a method for single cell stiffness measurement based on a nano-needle and nanomanipulation. The nano-needle with a buffering beam was fabricated from an atomic force microscope cantilever by the focused ion beam etching technique. Wild type yeast cells (W303) were prepared and placed on the sample stage inside an environmental scanning electron microscope (ESEM) chamber. The nanomanipulator actuated the nano-needle to press against a single yeast cell. As a result, the deformation of the cell and nano-needle was observed by the ESEM system in real-time. Finally, the stiffness of the single cell was determined based on this deformation information. To reveal the relationship between the cell stiffness and the environmental humidity conditions, the cell stiffness was measured at three different humidity conditions, i.e. 40, 70 and 100%, respectively. The results show that the stiffness of a single cell is reduced with increasing humidity.

  17. T cells conditioned with MDSC show an increased anti-tumor activity after adoptive T cell based immunotherapy.

    PubMed

    Raber, Patrick L; Sierra, Rosa A; Thevenot, Paul T; Shuzhong, Zhang; Wyczechowska, Dorota D; Kumai, Takumi; Celis, Esteban; Rodriguez, Paulo C

    2016-04-05

    The success of adoptive T cell-based immunotherapy (ACT) in cancer is limited in part by the accumulation of myeloid-derived suppressor cells (MDSC), which block several T cell functions, including T cell proliferation and the expression of various cytotoxic mediators. Paradoxically, the inhibition of CD8+ T cell differentiation into cytotoxic populations increased their efficacy after ACT into tumor-bearing hosts. Therefore, we aimed to test the impact of conditioning CD8+ T cells with MDSC on their differentiation potential and ACT efficacy. Our results indicate that MDSC impaired the progression of CD8+ T cells into effector populations, without altering their activation status, production of IL-2, or signaling through the T cell receptor. In addition, culture of CD8+ T cells with MDSC resulted in an increased ACT anti-tumor efficacy, which correlated with a higher frequency of the transferred T cells and elevated IFNγ production. Interestingly, activated CD62L+ CD8+ T cells were responsible for the enhanced anti-tumor activity showed by MDSC-exposed T cells. Additional results showed a decreased protein synthesis rate and lower activity of the mammalian/mechanistic target of rapamycin (mTOR) in T cells conditioned with MDSC. Silencing of the negative mTOR regulator tuberous sclerosis complex-2 in T cells co-cultured with MDSC restored mTOR activity, but resulted in T cell apoptosis. These results indicate that conditioning of T cells with MDSC induces stress survival pathways mediated by a blunted mTOR signaling, which regulated T cell differentiation and ACT efficacy. Continuation of this research will enable the development of better strategies to increase ACT responses in cancer.

  18. Alternative luciferase for monitoring bacterial cells under adverse conditions.

    PubMed

    Wiles, Siouxsie; Ferguson, Kathryn; Stefanidou, Martha; Young, Douglas B; Robertson, Brian D

    2005-07-01

    The availability of cloned luciferase genes from fireflies (luc) and from bacteria (luxAB) has led to the widespread use of bioluminescence as a reporter to measure cell viability and gene expression. The most commonly occurring bioluminescence system in nature is the deep-sea imidazolopyrazine bioluminescence system. Coelenterazine is an imidazolopyrazine derivative which, when oxidized by an appropriate luciferase enzyme, produces carbon dioxide, coelenteramide, and light. The luciferase from the marine copepod Gaussia princeps (Gluc) has recently been cloned. We expressed the Gluc gene in Mycobacterium smegmatis using a shuttle vector and compared its performance with that of an existing luxAB reporter. In contrast to luxAB, the Gluc luciferase retained its luminescence output in the stationary phase of growth and exhibited enhanced stability during exposure to low pH, hydrogen peroxide, and high temperature. The work presented here demonstrated the utility of the copepod luciferase bioluminescent reporter as an alternative to bacterial luciferase, particularly for monitoring responses to environmental stress stimuli.

  19. Standard culture medium allows clonal dilution of Trypanosoma brucei procyclic cells after auto-conditioning.

    PubMed

    Archer, Stuart K

    2009-03-01

    Trypanosoma brucei can be cultured in vitro in the mammalian bloodstream form or in the procyclic (PC) form found in the insect vector. Bloodstream trypanosomes can be cloned by limiting dilution, but PCs can only be diluted in conditioned medium, i.e., medium in which PC cells have previously been grown. It is shown here that this limitation does not apply to the most commonly used PC cell strain, Lister 427, if free radicals are removed from the medium. The reported benefit of conditioning media may arise in part from a process of hemin-catalysed depletion of peroxide ("auto-conditioning") which occurs during extended incubation at growth temperature. Scavenging free radicals by addition of pyruvate also improves PC cell viability. However, other PC cell strains such as Treu 927 require cell-conditioned media unless grown in a 5% CO2 atmosphere. Several other culture parameters that affect growth rates and dilution capability were identified.

  20. ReNCell VM conditioned medium enhances the induction of dental pulp stem cells into dopaminergic like cells.

    PubMed

    Gnanasegaran, Nareshwaran; Govindasamy, Vijayendran; Musa, Sabri; Abu Kasim, Noor Hayaty

    2016-03-01

    Among the debilitating diseases, neurological related diseases are the most challenging ones to be treated using cell replacement therapies. Recently, dental pulp stem cells (SHED) were found to be most suitable cell choice for neurological related diseases as evidenced with many preclinical studies. To enhance the neurological potential of SHED, we recapitulated one of the pharmacological therapeutic tools in cell replacement treatment, we pre-conditioned dental pulp stem cells (SHED) with culture medium of ReNCell VM, an immortalized neuron progenitor cell, prior to neurogenesis induction and investigated whether this practice enhances their neurogenesis potential especially towards dopaminergic neurons. We hypothesed that the integration of pharmacological practices such as co-administration of various drugs, a wide range of doses and duration as well as pre-conditioning into cell replacement may enhance the efficacy of stem cell therapy. In particular, pre-conditioning is shown to be involved in the protective effect from some membrano-tropic drugs, thereby improving the resistance of cell structures and homing capabilities. We found that cells pre-treated with ReNCell VM conditioned medium displayed bipolar structures with extensive branches resembling putative dopaminergic neurons as compared to non-treated cells. Furthermore, many neuronal related markers such as NES, NR4A2, MSI1, and TH were highly expressed (fold changes > 2; p < 0.05) in pre-treated cells. Similar observations were detected at the protein level. The results demonstrate for the first time that SHED pre-conditioning enhances neurological potential and we suggest that cells should be primed to their respective environment prior to transplantation.

  1. Differentiation of mouse induced pluripotent stem cells into neurons using conditioned medium of dorsal root ganglia.

    PubMed

    Kitazawa, Ayako; Shimizu, Norio

    2011-07-01

    Mouse induced pluripotent stem (iPS) cells are known to have the ability to differentiate into various cell lineages including neurons in vitro. We have reported that chick dorsal root ganglion (DRG)-conditioned medium (CM) promoted the differentiation of mouse embryonic stem (ES) cells into motor neurons. We investigated the formation of undifferentiated iPS cell colonies and the differentiation of iPS cells into neurons using DRG-CM. When iPS cells were cultured in DMEM containing leukemia inhibitory factor (LIF), the iPS cells appeared to be maintained in an undifferentiated state for 19 passages. The number of iPS cell colonies (200 μm in diameter) was maximal at six days of cultivation and the colonies were maintained in an undifferentiated state, but the iPS cell colonies at ten days of cultivation had hollows inside the colonies and were differentiated. By contrast, the number of ES cell colonies (200 μm in diameter) was maximal at ten days of cultivation. The iPS cells were able to proliferate and differentiate easily into various cell lineages, compared to ES cells. When iPS cell colonies were cultured in a manner similar to ES cells with DMEM/F-12K medium supplemented with DRG-CM, the iPS cells mainly differentiated into motor and sensory neurons. These results suggested that the differentiation properties of iPS cells differ from those of ES cells.

  2. Distinct patterns of cell motion inside a micro-channel under different osmotic conditions.

    PubMed

    Tsai, Chia-Hung Dylan; Kaneko, Makoto; Sakuma, Shinya; Arai, Fumihito

    2013-01-01

    The effect of osmotic condition on a living cell inside a micro-channel is firstly studied in this work. By utilizing a high-speed camera, we observed distinct patterns of cell motion under different osmotic conditions, which are established by saline with different concentrations of sodium chloride (NaCl). The cell motions are tracked by a computer, and are presented by the coordinates of location and time (x-t chart). The motions of cells under hypotonic condition (NaCl% < 0.9%) are convex curves on the chart while the ones under isotonic and hypertonic conditions (NaCl% ≥ 0.9%) are concave curves. Since saline is widely used in both medical practices and cell-related researches, our results point out two important facts: 1) Cells are sensitive to the percentage of NaCl. One percent difference in overall concentration makes dramatic changes in cell characteristics, such as cell stiffness. 2) The micro-channel method can clearly tell the difference between hypotonic, isotonic and hypertonic conditions according to the pattern of cell motion. Interpretations of the phenomena from different perspectives are also discussed in this paper.

  3. Cellular interactions via conditioned media induce in vivo nephron generation from tubular epithelial cells or mesenchymal stem cells

    SciTech Connect

    Machiguchi, Toshihiko Nakamura, Tatsuo

    2013-06-07

    Highlights: •We have attempted in vivo nephron generation using conditioned media. •Vascular and tubular cells do cross-talks on cell proliferation and tubular changes. •Tubular cells suppress these changes in mesenchymal stem cells. •Tubular cells differentiate mesenchymal stem cells into tubular cells. •Nephrons can be created from implanted tubular cells or mesenchymal stem cells. -- Abstract: There are some successful reports of kidney generation by utilizing the natural course of kidney development, namely, the use of an artificially treated metanephros, blastocyst or ureteric bud. Under a novel concept of cellular interactions via conditioned media (CMs), we have attempted in vivo nephron generation from tubular epithelial cells (TECs) or mesenchymal stem cells (MSCs). Here we used 10× CMs of vascular endothelial cells (VECs) and TECs, which is the first to introduce a CM into the field of organ regeneration. We first present stimulative cross-talks induced by these CMs between VECs and TECs on cell proliferation and morphological changes. In MSCs, TEC-CM suppressed these changes, however, induced cytokeratin expression, indicating the differentiation of MSCs into TECs. As a result, glomerular and tubular structures were created following the implantation of TECs or MSCs with both CMs. Our findings suggest that the cellular interactions via CMs might induce in vivo nephron generation from TECs or MSCs. As a promoting factor, CMs could also be applied to the regeneration of other organs and tissues.

  4. Synapses lacking astrocyte appear in the amygdala during consolidation of Pavlovian threat conditioning.

    PubMed

    Ostroff, Linnaea E; Manzur, Mustfa K; Cain, Christopher K; Ledoux, Joseph E

    2014-06-15

    There is growing evidence that astrocytes, long held to merely provide metabolic support in the adult brain, participate in both synaptic plasticity and learning and memory. Astrocytic processes are sometimes present at the synaptic cleft, suggesting that they might act directly at individual synapses. Associative learning induces synaptic plasticity and morphological changes at synapses in the lateral amygdala (LA). To determine whether astrocytic contacts are involved in these changes, we examined LA synapses after either threat conditioning (also called fear conditioning) or conditioned inhibition in adult rats by using serial section transmission electron microscopy (ssTEM) reconstructions. There was a transient increase in the density of synapses with no astrocytic contact after threat conditioning, especially on enlarged spines containing both polyribosomes and a spine apparatus. In contrast, synapses with astrocytic contacts were smaller after conditioned inhibition. This suggests that during memory consolidation astrocytic processes are absent if synapses are enlarging but present if they are shrinking. We measured the perimeter of each synapse and its degree of astrocyte coverage, and found that only about 20-30% of each synapse was ensheathed. The amount of synapse perimeter surrounded by astrocyte did not scale with synapse size, giving large synapses a disproportionately long astrocyte-free perimeter and resulting in a net increase in astrocyte-free perimeter after threat conditioning. Thus astrocytic processes do not mechanically isolate LA synapses, but may instead interact through local signaling, possibly via cell-surface receptors. Our results suggest that contact with astrocytic processes opposes synapse growth during memory consolidation.

  5. The use of stem cells for the treatment of spinal surgical conditions.

    PubMed

    Aftab, Syed; Chimutengwende-Gordon, Mukai; Malik, Atif; Lee, Robert

    2013-11-01

    Spinal pathologies are a major burden on society and individuals. Recent years have seen a large number of studies dedicated to the use of stem cells in spinal surgery. This review focuses on recent advances and controversies regarding the applications of stem cells in spinal fusion surgery, spinal cord injury and intervertebral disc degeneration. There are significant concerns regarding the ethics and risks of stem cell use. Animal models do not always accurately depict the human condition. While a great deal has been achieved, successful translation into clinical practice is needed. However there is no doubt that stem cells have a major role to play in the future management of spinal conditions.

  6. Metformin increases PDH and suppresses HIF-1α under hypoxic conditions and induces cell death in oral squamous cell carcinoma

    PubMed Central

    Guimarães, Talita Antunes; Farias, Lucyana Conceição; Santos, Eliane Sobrinho; de Carvalho Fraga, Carlos Alberto; Orsini, Lissur Azevedo; de Freitas Teles, Leandro; Feltenberger, John David; de Jesus, Sabrina Ferreira; de Souza, Marcela Gonçalves; Sousa Santos, Sérgio Henrique; de Paula, Alfredo Maurício Batista

    2016-01-01

    Background Metformin is a biguanide, belonging to the oral hypoglycemic agents and is a widely used in the treatment of type 2 diabetes. Evidence indicate that Metformin inhibits cell proliferation in several human cancers and inhibits the Warburg phenomenon in tumor cells. Results Low PDH levels were observed in OSCC, and Metformin promotes an increase in PDH levels in hypoxic conditions. Metformin also reduced HIF-1α mRNA and protein levels. Metformin demonstrated antiproliferative effects, inhibited migration, increased the number of apoptotic cells and increased the transcription of caspase 3. Objective The present study aims to explore the effects of Metformin in hypoxic conditions. Specifically, we focused on pyruvate dehydrogenase (PDH), (hypoxia-inducible factor 1α) HIF-1α levels and the oral squamous cell carcinoma (OSCC) cell phenotype. Additionally, we also investigated a theoretical consequence of Metformin treatment. Methods PDH levels in patients with OSCC and oral dysplasia were evaluated. Metformin was administered in vitro to test the effect of Metformin under hypoxic conditions. The results were complemented by Bioinformatics analyses. Conclusions In conclusion, our current findings show that Metformin reduces HIF-1α gene expression and increases PDH expression. Metformin inhibits cell proliferation and migration in the OSCC cell line model. Additionally, Metformin enhances the number of apoptotic cells and caspase 3 levels. Interestingly enough, Metformin did not increase the mutant p53 levels under hypoxic conditions. PMID:27474170

  7. Conditioned medium as a strategy for human stem cells chondrogenic differentiation.

    PubMed

    Alves da Silva, M L; Costa-Pinto, A R; Martins, A; Correlo, V M; Sol, P; Bhattacharya, M; Faria, S; Reis, R L; Neves, Nuno M

    2015-06-01

    Paracrine signalling from chondrocytes has been reported to increase the synthesis and expression of cartilage extracellular matrix (ECM) by stem cells. The use of conditioned medium obtained from chondrocytes for stimulating stem cells chondrogenic differentiation may be a very interesting alternative for moving into the clinical application of these cells, as chondrocytes could be partially replaced by stem cells for this type of application. In the present study we aimed to achieve chondrogenic differentiation of two different sources of stem cells using conditioned medium, without adding growth factors. We tested both human bone marrow-derived mesenchymal stem cells (hBSMCs) and human Wharton's jelly-derived stem cells (hWJSCs). Conditioned medium obtained from a culture of human articular chondrocytes was used to feed the cells during the experiment. Cultures were performed in previously produced three-dimensional (3D) scaffolds, composed of a blend of 50:50 chitosan:poly(butylene succinate). Both types of stem cells were able to undergo chondrogenic differentiation without the addition of growth factors. Cultures using hWJSCs showed significantly higher GAGs accumulation and expression of cartilage-related genes (aggrecan, Sox9 and collagen type II) when compared to hBMSCs cultures. Conditioned medium obtained from articular chondrocytes induced the chondrogenic differentiation of MSCs and ECM formation. Obtained results showed that this new strategy is very interesting and should be further explored for clinical applications.

  8. Vascular endothelial cells in cell-mediated immunity: adoptive transfer with in vitro conditioned cells is genetically restricted at the endothelial cell barrier

    SciTech Connect

    Standage, B.A.; Vetto, R.M.; Jones, R.; Burger, D.R.

    1985-01-01

    Delayed-type hypersensitivity (DTH) is a cell-mediated immune response that can be adoptively transferred in rats when greater than 2 X 10(8) cells from peritoneal exudate, lymph nodes, or spleen are used. We have shown that by using an in vitro conditioning step with antigen, transfer can be subsequently carried out with as few as 2 X 10(7) spleen cells. The magnitude of DTH was reflected in ear swelling after intradermal injection of antigen (tuberculin or keyhole limpet hemocyanin (KLH)) and confirmed histologically. The transfer was antigen specific, requiring the sensitizing antigen in both the in vitro conditioning step and in the ear test challenge. Adoptive transfer with conditioned cells was genetically restricted by alleles of the RT-1 region (major histocompatibility complex (MHC) of the rat). Brown Norway strain (n haplotype) immune cells would not transfer DTH to Lewis (1 haplotype), ACI (a haplotype), or Buffalo (b haplotype) rats, whereas each strain would transfer DTH to syngeneic recipients. Moreover, this pattern of restriction held for all strains when tested in reciprocal fashion. In additional experiments, F1 to parental bone marrow chimeras were constructed so that bone-marrow-derived cells and non-bone-marrow-derived cells were of different RT-1 haplotypes. When these chimeras were used as recipients, transfer of DTH was only observed when immune donor cells and recipient non-bone-marrow-derived cells were syngeneic. These results point to the critical role of non-bone-marrow-derived cells (endothelial cells) in the DTH reaction.

  9. Centrosome misorientation mediates slowing of the cell cycle under limited nutrient conditions in Drosophila male germline stem cells.

    PubMed

    Roth, Therese M; Chiang, C-Y Ason; Inaba, Mayu; Yuan, Hebao; Salzmann, Viktoria; Roth, Caitlin E; Yamashita, Yukiko M

    2012-04-01

    Drosophila male germline stem cells (GSCs) divide asymmetrically, balancing self-renewal and differentiation. Although asymmetric stem cell division balances between self-renewal and differentiation, it does not dictate how frequently differentiating cells must be produced. In male GSCs, asymmetric GSC division is achieved by stereotyped positioning of the centrosome with respect to the stem cell niche. Recently we showed that the centrosome orientation checkpoint monitors the correct centrosome orientation to ensure an asymmetric outcome of the GSC division. When GSC centrosomes are not correctly oriented with respect to the niche, GSC cell cycle is arrested/delayed until the correct centrosome orientation is reacquired. Here we show that induction of centrosome misorientation upon culture in poor nutrient conditions mediates slowing of GSC cell proliferation via activation of the centrosome orientation checkpoint. Consistently, inactivation of the centrosome orientation checkpoint leads to lack of cell cycle slowdown even under poor nutrient conditions. We propose that centrosome misorientation serves as a mediator that transduces nutrient information into stem cell proliferation, providing a previously unappreciated mechanism of stem cell regulation in response to nutrient conditions.

  10. Tumor-associated fibroblast-conditioned medium induces CDDP resistance in HNSCC cells

    PubMed Central

    Steinbichler, Teresa Bernadette; Metzler, Veronika; Pritz, Christian; Riechelmann, Herbert; Dudas, Jozsef

    2016-01-01

    Objective EMT (epithelial to mesenchymal transition) contributes to tumor progression and metastasis. We aimed to investigate the effects of EMT on CDDP resistance in HNSCC (head and neck squamous cell carcinoma)-cells. Methods EMT was induced using conditioned medium from a tumor cell/fibroblast co-culture. HNSCC cells were alternatively treated with TGF-β1. The response to CDDP was evaluated with viability and clonogenic assays. Results Treatment of SCC-25/Detroit 562 cells with conditioned medium increased viability of the tumor cells. Moreover, it doubled the IC50 of CDDP of SCC-25 cells from 6.2 μM to 13.1 μM (p < 0.001). The IC50 of CDDP of Detroit 562 cells was increased following treatment with conditioned medium from 13.1 μM to 26.8 μM (p < 0.01). Colony forming ability after treatment with 5 or 10 μM CDDP was significantly higher in HNSCC cells treated with co-culture conditioned medium than in controls (p < 0.05). Treatment with TGF-β1 had no effect on the IC50 of CDDP (p > 0.1). Conclusions Cell free medium from a co-culture was able to induce EMT in HNSCC cells. Co-culture treated HNSCC cells revealed increased viability and were less sensitive to CDDP treatment. TGF-β1 also induced a mesenchymal phenotype, but did not alter resistance to CDDP in HNSCC cells. PMID:26497215

  11. Eosinophil adhesion under flow conditions activates mechanosensitive signaling pathways in human endothelial cells

    PubMed Central

    Cuvelier, Susan L.; Paul, Smitha; Shariat, Neda; Colarusso, Pina; Patel, Kamala D.

    2005-01-01

    Leukocyte transmigration can be affected by shear stress; however, the mechanisms by which shear stress modulates transmigration are unknown. We found that adhesion of eosinophils or an eosinophilic cell line to intereukin 4–stimulated endothelial cells led to a shear-dependent increase in endothelial cell intracellular calcium and increased phosphorylation of extracellular signal-regulated kinase (ERK) 2, but not c-Jun NH2-terminal kinase or p38 mitogen-activated protein kinase. Latex beads coated with antibodies were used to characterize the role of specific endothelial cell surface molecules in initiating signaling under shear conditions. We found that ligation of either vascular cell adhesion molecule–1 or E-selectin, but not major histocompatibility complex class I, induced a shear-dependent increase in ERK2 phosphorylation in cytokine-stimulated endothelial cells. Disassembly of the actin cytoskeleton with latrunculin A prevented ERK2 phosphorylation after adhesion under flow conditions, supporting a role for the cytoskeleton in mechanosensing. Rapid phosphorylation of focal adhesion kinase and paxillin occurred under identical conditions, suggesting that focal adhesions were also involved in mechanotransduction. Finally, we found that Rho-associated protein kinase and calpain were both critical in the subsequent transendothelial migration of eosinophils under flow conditions. These data suggest that ligation of leukocyte adhesion molecules under flow conditions leads to mechanotransduction in endothelial cells, which can regulate subsequent leukocyte trafficking. PMID:16172263

  12. Eosinophil adhesion under flow conditions activates mechanosensitive signaling pathways in human endothelial cells.

    PubMed

    Cuvelier, Susan L; Paul, Smitha; Shariat, Neda; Colarusso, Pina; Patel, Kamala D

    2005-09-19

    Leukocyte transmigration can be affected by shear stress; however, the mechanisms by which shear stress modulates transmigration are unknown. We found that adhesion of eosinophils or an eosinophilic cell line to intereukin 4-stimulated endothelial cells led to a shear-dependent increase in endothelial cell intracellular calcium and increased phosphorylation of extracellular signal-regulated kinase (ERK) 2, but not c-Jun NH2-terminal kinase or p38 mitogen-activated protein kinase. Latex beads coated with antibodies were used to characterize the role of specific endothelial cell surface molecules in initiating signaling under shear conditions. We found that ligation of either vascular cell adhesion molecule-1 or E-selectin, but not major histocompatibility complex class I, induced a shear-dependent increase in ERK2 phosphorylation in cytokine-stimulated endothelial cells. Disassembly of the actin cytoskeleton with latrunculin A prevented ERK2 phosphorylation after adhesion under flow conditions, supporting a role for the cytoskeleton in mechano-sensing. Rapid phosphorylation of focal adhesion kinase and paxillin occurred under identical conditions, suggesting that focal adhesions were also involved in mechanotransduction. Finally, we found that Rho-associated protein kinase and calpain were both critical in the subsequent transendothelial migration of eosinophils under flow conditions. These data suggest that ligation of leukocyte adhesion molecules under flow conditions leads to mechanotransduction in endothelial cells, which can regulate subsequent leukocyte trafficking.

  13. Non-genotoxic conditioning for hematopoietic stem cell transplantation using a hematopoietic-cell-specific internalizing immunotoxin

    PubMed Central

    Palchaudhuri, Rahul; Saez, Borja; Hoggatt, Jonathan; Schajnovitz, Amir; Sykes, David B; Tate, Tiffany A; Czechowicz, Agnieszka; Kfoury, Youmna; Ruchika, FNU; Rossi, Derrick J; Verdine, Gregory L; Mansour, Michael K; Scadden, David T

    2016-01-01

    Hematopoietic stem cell transplantation (HSCT) offers curative therapy for patients with hemoglobinopathies, congenital immunodeficiencies, and other conditions, possibly including AIDS. Autologous HSCT using genetically corrected cells would avoid the risk of graft-versus-host disease (GVHD), but the genotoxicity of conditioning remains a substantial barrier to the development of this approach. Here we report an internalizing immunotoxin targeting the hematopoietic-cell-restricted CD45 receptor that effectively conditions immunocompetent mice. A single dose of the immunotoxin, CD45–saporin (SAP), enabled efficient (>90%) engraftment of donor cells and full correction of a sickle-cell anemia model. In contrast to irradiation, CD45–SAP completely avoided neutropenia and anemia, spared bone marrow and thymic niches, enabling rapid recovery of T and B cells, preserved anti-fungal immunity, and had minimal overall toxicity. This non-genotoxic conditioning method may provide an attractive alternative to current conditioning regimens for HSCT in the treatment of non-malignant blood diseases. PMID:27272386

  14. Blood Stem Cell Activity Is Arrested by Th1-Mediated Injury Preventing Engraftment following Nonmyeloablative Conditioning

    PubMed Central

    Florek, Mareike; Kohrt, Holbrook E. K.; Küpper, Natascha J.; Filatenkov, Alexander; Linderman, Jessica A.; Hadeiba, Husein; Negrin, Robert S.

    2016-01-01

    T cells are widely used to promote engraftment of hematopoietic stem cells (HSCs) during an allogeneic hematopoietic cell transplantation. Their role in overcoming barriers to HSC engraftment is thought to be particularly critical when patients receive reduced doses of preparative chemotherapy and/or radiation compared with standard transplantations. In this study, we sought to delineate the effects CD4+ cells on engraftment and blood formation in a model that simulates clinical hematopoietic cell transplantation by transplanting MHC-matched, minor histocompatibility–mismatched grafts composed of purified HSCs, HSCs plus bulk T cells, or HSCs plus T cell subsets into mice conditioned with low-dose irradiation. Grafts containing conventional CD4+ T cells caused marrow inflammation and inhibited HSC engraftment and blood formation. Posttransplantation, the marrows of HSCs plus CD4+ cell recipients contained IL-12–secreting CD11c+ cells and IFN-γ–expressing donor Th1 cells. In this setting, host HSCs arrested at the short-term stem cell stage and remained in the marrow in a quiescent cell cycling state (G0). As a consequence, donor HSCs failed to engraft and hematopoiesis was suppressed. Our data show that Th1 cells included in a hematopoietic allograft can negatively impact HSC activity, blood reconstitution, and engraftment of donor HSCs. This potential negative effect of donor T cells is not considered in clinical transplantation in which bulk T cells are transplanted. Our findings shed new light on the effects of CD4+ T cells on HSC biology and are applicable to other pathogenic states in which immune activation in the bone marrow occurs such as aplastic anemia and certain infectious conditions. PMID:27815446

  15. RSS1 regulates the cell cycle and maintains meristematic activity under stress conditions in rice

    PubMed Central

    Ogawa, Daisuke; Abe, Kiyomi; Miyao, Akio; Kojima, Mikiko; Sakakibara, Hitoshi; Mizutani, Megumi; Morita, Haruka; Toda, Yosuke; Hobo, Tokunori; Sato, Yutaka; Hattori, Tsukaho; Hirochika, Hirohiko; Takeda, Shin

    2011-01-01

    Plant growth and development are sustained by continuous cell division in the meristems, which is perturbed by various environmental stresses. For the maintenance of meristematic functions, it is essential that cell division be coordinated with cell differentiation. However, it is unknown how the proliferative activities of the meristems and the coordination between cell division and differentiation are maintained under stressful conditions. Here we show that a rice protein, RSS1, whose stability is controlled by cell cycle phases, contributes to the vigour of meristematic cells and viability under salinity conditions. These effects of RSS1 are exerted by regulating the G1–S transition, possibly through an interaction of RSS1 with protein phosphatase 1, and are mediated by the phytohormone, cytokinin. RSS1 is conserved widely in plant lineages, except eudicots, suggesting that RSS1-dependent mechanisms might have been adopted in specific lineages during the evolutionary radiation of angiosperms. PMID:21505434

  16. Characterization and optimization of cathodic conditions for H2O2 synthesis in microbial electrochemical cells

    EPA Science Inventory

    Cathode potential and O2 supply methods were investigated to improve H2O2 synthesis in an electrochemical cell, and optimal cathode conditions were applied for microbial electrochemical cells (MECs). Using aqueous O2 for the cathode significantly improved current density, but H2...

  17. Purkinje Cell Activity in the Cerebellar Anterior Lobe after Rabbit Eyeblink Conditioning

    ERIC Educational Resources Information Center

    Green, John T.; Steinmetz, Joseph E.

    2005-01-01

    The cerebellar anterior lobe may play a critical role in the execution and proper timing of learned responses. The current study was designed to monitor Purkinje cell activity in the rabbit cerebellar anterior lobe after eyeblink conditioning, and to assess whether Purkinje cells in recording locations may project to the interpositus nucleus.…

  18. Concise Review: Stem Cell Trials Using Companion Animal Disease Models.

    PubMed

    Hoffman, Andrew M; Dow, Steven W

    2016-07-01

    Studies to evaluate the therapeutic potential of stem cells in humans would benefit from more realistic animal models. In veterinary medicine, companion animals naturally develop many diseases that resemble human conditions, therefore, representing a novel source of preclinical models. To understand how companion animal disease models are being studied for this purpose, we reviewed the literature between 2008 and 2015 for reports on stem cell therapies in dogs and cats, excluding laboratory animals, induced disease models, cancer, and case reports. Disease models included osteoarthritis, intervertebral disc degeneration, dilated cardiomyopathy, inflammatory bowel diseases, Crohn's fistulas, meningoencephalomyelitis (multiple sclerosis-like), keratoconjunctivitis sicca (Sjogren's syndrome-like), atopic dermatitis, and chronic (end-stage) kidney disease. Stem cells evaluated in these studies included mesenchymal stem-stromal cells (MSC, 17/19 trials), olfactory ensheathing cells (OEC, 1 trial), or neural lineage cells derived from bone marrow MSC (1 trial), and 16/19 studies were performed in dogs. The MSC studies (13/17) used adipose tissue-derived MSC from either allogeneic (8/13) or autologous (5/13) sources. The majority of studies were open label, uncontrolled studies. Endpoints and protocols were feasible, and the stem cell therapies were reportedly safe and elicited beneficial patient responses in all but two of the trials. In conclusion, companion animals with naturally occurring diseases analogous to human conditions can be recruited into clinical trials and provide realistic insight into feasibility, safety, and biologic activity of novel stem cell therapies. However, improvements in the rigor of manufacturing, study design, and regulatory compliance will be needed to better utilize these models. Stem Cells 2016;34:1709-1729.

  19. A review of accelerated conditioning for a polymer electrolyte membrane fuel cell

    NASA Astrophysics Data System (ADS)

    Yuan, Xiao-Zi; Zhang, Shengsheng; Sun, Jian Colin; Wang, Haijiang

    A newly fabricated polymer electrolyte membrane (PEM) fuel cell usually needs a so-called break-in/conditioning/incubation period to activate it and reach its best performance. Typically, during this activation period the cell performance increases gradually, and then reaches a plateau without further increase. Depending on the membrane electrode assemblies, this process can take hours and even days to complete, which consumes a considerable amount of hydrogen fuel, leading to a higher operating cost. To provide for accelerated conditioning techniques that can complete the process in a short time period, this paper reviews established conditioning protocols and reported methods to condition PEM single cells and stacks, in an attempt to summarize available information on PEM fuel cell conditioning and the underlying mechanisms. Various techniques are arranged into two categories: on-line conditioning and off-line conditioning. For each technique, the experimental procedure and outcomes are outlined. Finally, weaknesses of the currently used conditioning techniques are indicated and further research efforts are proposed.

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

    PubMed

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

    1977-12-01

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

  1. An integrated system for synchronous culture of animal cells under controlled conditions.

    PubMed

    Mendoza-Pérez, Elena; Hernández, Vanessa; Palomares, Laura A; Serrato, José A

    2016-01-01

    The cell cycle has fundamental effects on cell cultures and their products. Tools to synchronize cultured cells allow the study of cellular physiology and metabolism at particular cell cycle phases. However, cells are most often arrested by methods that alter their homeostasis and are then cultivated in poorly controlled environments. Cell behavior could then be affected by the synchronization method and culture conditions used, and not just by the particular cell cycle phase under study. Moreover, only a few viable cells are recovered. Here, we designed an integrated system where a large number of cells from a controlled bioreactor culture is separated by centrifugal elutriation at high viabilities. In contrast to current elutriation methods, cells are injected directly from a bioreactor into an injection loop, allowing the introduction of a large number of cells into the separation chamber without stressful centrifugation. A low pulsation peristaltic pump increases the stability of the elutriation chamber. Using this approach, a large number of healthy cells at each cell cycle phase were obtained, allowing their direct inoculation into fully instrumented bioreactors. Hybridoma cells synchronized and cultured in this system behaved as expected for a synchronous culture.

  2. Viable Mononuclear Cell Stability Study for Implementation in a Proficiency Testing Program: Impact of Shipment Conditions

    PubMed Central

    Davis, Kristine; Glazer, Barbara; De Souza, Yvonne; Kessler, Joseph; Betsou, Fotini

    2014-01-01

    The impact of shipping temperatures and preservation media used during transport of either peripheral blood mononuclear cells (PBMCs) or Jurkat cells was assessed, in view of implementing of a proficiency testing scheme on mononuclear cell viability. Samples were analyzed before and after shipment at different temperatures (ambient temperature, dry ice, and liquid nitrogen) and in different preservation media (serum with cryoprotectant, commercial cryopreservation solution, and room temperature transport medium). Sample quality was assessed by viability assays (Trypan Blue dye exclusion, flow cytometry, Cell Analysis System cell counting (CASY)), and by ELISpot functional assay. The liquid nitrogen storage and shipment were found to be the most stable conditions to preserve cell viability and functionality. However, we show that alternative high quality shipment conditions for viable cells are dry ice shipment and commercial cryopreservation solution. These were also cost-efficient shipment conditions, satisfying the requirements of a proficiency testing scheme for viable mononuclear cells. Room temperature transport medium dramatically and adversely affected the integrity of mononuclear cells. PMID:24955735

  3. Raman spectroscopy of a single living cell in environmentally stressed conditions

    NASA Astrophysics Data System (ADS)

    Singh, Gajendra P.; Creely, Caitriona; Volpe, Giovanni; Grotsch, Helga; Petrov, Dmitri

    2005-08-01

    Living cells initiate a stress response in order to survive environmentally stressful conditions. We monitored changes in the Raman spectra of an optically trapped Saccharomyces cerevisiae yeast cell under normal and hyperosmotic stress conditions. When the yeast cells were challenged with a high concentration of glucose so as to exert hyperosmotic stress, it was shown that two chemical substances - glycerol and ethanol - could be monitored in real time in a single cell. The volume of the detection area of our confocal microspectrometer is approximately 1 fL. The average quantities of detected glycerol and ethanol are about 300 attomol and 700 attomol respectively. This amounts to the detection of approximately 108 glycerol molecules and 4 X 108 ethanol molecules after 36 min of hyper osmotic stress. Besides this, we also optically trapped a single yeast cell for up to three hours under normal conditions and monitored the changes in the Raman spectra during the lag phase of its growth and the G1 phase of its cell cycle. During the lag phase the cell synthesises new proteins and the observed behavior of the peaks corresponding to these proteins as well as those of RNA served as a sensitive indicator of the adaptation of the cell to its changed environment. The changes observed in the Raman spectra of a trapped yeast cell in the late G1 phase or the beginning of S phase corresponded to the growth of a bud.

  4. Hair Regeneration Treatment Using Adipose-Derived Stem Cell Conditioned Medium: Follow-up With Trichograms

    PubMed Central

    Suga, Hirotaka

    2015-01-01

    Objective: Adipose-derived stem cells secrete various growth factors that promote hair growth. This study examined the effects of adipose-derived stem cell-conditioned medium on alopecia. Methods: Adipose-derived stem cell-conditioned medium was intradermally injected in 22 patients (11 men and 11 women) with alopecia. Patients received treatment every 3 to 5 weeks for a total of 6 sessions. Hair numbers were counted using trichograms before and after treatment. A half-side comparison study was also performed in 10 patients (8 men and 2 women). Results: Hair numbers were significantly increased after treatment in both male (including those without finasteride administration) and female patients. In the half-side comparison study, the increase in hair numbers was significantly higher on the treatment side than on the placebo side. Conclusion: Treatment using adipose-derived stem cell-conditioned medium appears highly effective for alopecia and may represent a new therapy for hair regeneration. PMID:25834689

  5. Maintenance of human embryonic stem cells in animal serum- and feeder layer-free culture conditions.

    PubMed

    Amit, Michal; Itskovitz-Eldor, Joseph

    2006-01-01

    The availability of human embryonic stem cells (hESCs) reflects their outstanding potential for research areas such as human developmental biology, teratology, and cell-based therapies. To allow their continuous growth as undifferentiated cells, isolation and culturing were traditionally conducted on mouse embryonic fibroblast feeder layers, using medium supplemented with fetal bovine serum. However, these conditions allow possible exposure of the cells to animal pathogens. Because both research and future clinical application require an animal-free and well-defined culture system for hESCs, these conventional conditions would prevent the use of hESCs in human therapy. This chapter describes optional culture conditions based on either animal-free or feeder-free culture methods for hESCs.

  6. Purkinje cell activity in the cerebellar anterior lobe after rabbit eyeblink conditioning

    PubMed Central

    Green, John T.; Steinmetz, Joseph E.

    2005-01-01

    The cerebellar anterior lobe may play a critical role in the execution and proper timing of learned responses. The current study was designed to monitor Purkinje cell activity in the rabbit cerebellar anterior lobe after eyeblink conditioning, and to assess whether Purkinje cells in recording locations may project to the interpositus nucleus. Rabbits were trained in an interstimulus interval discrimination procedure in which one tone signaled a 250-msec conditioned stimulus-unconditioned stimulus (CS-US) interval and a second tone signaled a 750-msec CS-US interval. All rabbits showed conditioned responses to each CS with mean onset and peak latencies that coincided with the CS-US interval. Many anterior lobe Purkinje cells showed significant learning-related activity after eyeblink conditioning to one or both of the CSs. More Purkinje cells responded with inhibition than with excitation to CS presentation. In addition, when the firing patterns of all conditioning-related Purkinje cells were pooled, it appeared that the population showed a pattern of excitation followed by inhibition during the CS-US interval. Using cholera toxin-conjugated horseradish peroxidase, Purkinje cells in recording areas were found to project to the interpositus nucleus. These data support previous studies that have suggested a role for the anterior cerebellar cortex in eyeblink conditioning as well as models of cerebellar-mediated CR timing that postulate that Purkinje cell activity inhibits conditioned response (CR) generation during the early portion of a trial by inhibiting the deep cerebellar nuclei and permits CR generation during the later portion of a trial through disinhibition of the cerebellar nuclei. PMID:15897252

  7. Microfluidically supported biochip design for culture of endothelial cell layers with improved perfusion conditions.

    PubMed

    Raasch, Martin; Rennert, Knut; Jahn, Tobias; Peters, Sven; Henkel, Thomas; Huber, Otmar; Schulz, Ingo; Becker, Holger; Lorkowski, Stefan; Funke, Harald; Mosig, Alexander

    2015-03-02

    Hemodynamic forces generated by the blood flow are of central importance for the function of endothelial cells (ECs), which form a biologically active cellular monolayer in blood vessels and serve as a selective barrier for macromolecular permeability. Mechanical stimulation of the endothelial monolayer induces morphological remodeling in its cytoskeleton. For in vitro studies on EC biology culture devices are desirable that simulate conditions of flow in blood vessels and allow flow-based adhesion/permeability assays under optimal perfusion conditions. With this aim we designed a biochip comprising a perfusable membrane that serves as cell culture platform multi-organ-tissue-flow (MOTiF biochip). This biochip allows an effective supply with nutrition medium, discharge of catabolic cell metabolites and defined application of shear stress to ECs under laminar flow conditions. To characterize EC layers cultured in the MOTiF biochip we investigated cell viability, expression of EC marker proteins and cell adhesion molecules of ECs dynamically cultured under low and high shear stress, and compared them with an endothelial culture in established two-dimensionally perfused flow chambers and under static conditions. We show that ECs cultured in the MOTiF biochip form a tight EC monolayer with increased cellular density, enhanced cell layer thickness, presumably as the result of a rapid and effective adaption to shear stress by remodeling of the cytoskeleton. Moreover, endothelial layers in the MOTiF biochip express higher amounts of EC marker proteins von-Willebrand-factor and PECAM-1. EC layers were highly responsive to stimulation with TNFα as detected at the level of ICAM-1, VCAM-1 and E-selectin expression and modulation of endothelial permeability in response to TNFα/IFNγ treatment under flow conditions. Compared to static and two-dimensionally perfused cell culture condition we consider MOTiF biochips as a valuable tool for studying EC biology in vitro under

  8. Selective Conditions Are Required for the Induction of Invariant NKT Cell Hyporesponsiveness by Antigenic Stimulation.

    PubMed

    Wingender, Gerhard; Birkholz, Alysia M; Sag, Duygu; Farber, Elisa; Chitale, Sampada; Howell, Amy R; Kronenberg, Mitchell

    2015-10-15

    Activation of invariant (i)NKT cells with the model Ag α-galactosylceramide induces rapid production of multiple cytokines, impacting a wide variety of different immune reactions. In contrast, following secondary activation with α-galactosylceramide, the behavior of iNKT cells is altered for months, with the production of most cytokines being strongly reduced. The requirements for the induction of this hyporesponsive state, however, remain poorly defined. In this study, we show that Th1-biasing iNKT cell Ags could induce iNKT cell hyporesponsiveness, as long as a minimum antigenic affinity was reached. In contrast, the Th2-biasing Ag OCH did not induce a hyporesponsive state, nor did cytokine-driven iNKT cell activation by LPS or infections. Furthermore, although dendritic cells and B cells have been reported to be essential for iNKT cell stimulation, neither dendritic cells nor B cells were required to induce iNKT cell hyporesponsiveness. Therefore, our data indicate that whereas some bone marrow-derived cells could induce iNKT cell hyporesponsiveness, selective conditions, dependent on the structure and potency of the Ag, were required to induce hyporesponsiveness.

  9. B-cell reconstitution for SCID: should a conditioning regimen be used in SCID treatment?

    PubMed

    Haddad, Elie; Leroy, Sandrine; Buckley, Rebecca H

    2013-04-01

    Bone marrow transplantation has resulted in life-saving sustained T-cell reconstitution in many infants with severe combined immunodeficiency (SCID), but correction of B-cell function has been more problematic. At the annual meeting of the Primary Immunodeficiency Treatment Consortium held in Boston, Massachusetts, on April 27, 2012, a debate was held regarding the use of pretransplantation conditioning versus no pretransplantation conditioning in an effort to address this problem. Reviews of the literature were made by both debaters, and there was agreement that there was a higher rate of B-cell chimerism and a lower number of patients who required ongoing immunoglobulin replacement therapy in centers that used pretransplantation conditioning. However, there were still patients who required immunoglobulin replacement in those centers, and therefore pretransplantation conditioning did not guarantee development of B-cell function. Dr Rebecca H. Buckley presented data on B-cell function according to the molecular defect of the patient, and showed that patients with IL-7 receptor α, ADA, and CD3 chain gene mutations can have normal B-cell function after transplantation with only host B cells. Dr Elie Haddad presented a statistical analysis of B-cell function in published reports and showed that only a conditioning regimen that contained busulfan was significantly associated with better B-cell function after transplantation. The question is whether the risk of immediate and long-term toxicity with use of busulfan is justified, particularly in patients with SCID with DNA repair defects and in very young newborns with SCID who will be detected by using newborn screening.

  10. Different Culture Conditions Modulate the Immunological Properties of Adipose Stem Cells

    PubMed Central

    Sivula, Jyrki; Huhtala, Heini; Helminen, Mika; Salo, Fanny; Mannerström, Bettina; Miettinen, Susanna

    2014-01-01

    The potential of human adipose stem cells (ASCs) for regenerative medicine has received recognition owing to their ease of isolation and their multilineage differentiation capacity. Additionally, low immunogenicity and immunosuppressive properties make them a relevant cell source when considering immunomodulation therapies and allogeneic stem cell treatments. In the current study, immunogenicity and immunosuppression of ASCs were determined through mixed lymphocyte reactions. The immunogenic response was analyzed after cell isolation and expansion in fetal bovine serum (FBS), human serum (HS)-supplemented medium, and xeno-free and serum-free (XF/SF) conditions. Additionally, the immunophenotype and the secretion of CXC chemokine ligand 8 (CXCL8), CXCL9, CXCL10, C-C chemokine ligand 2 (CCL2), CCL5, interleukin 2 (IL-2), IL-4, IL-6, IL-10, IL-17A, tumor necrosis factor-α, interferon-γ, transforming growth factor-β1, indoleamine 2,3-deoxygenase, Galectin-1, and Galectin-3 were analyzed. The results showed that ASCs were weakly immunogenic when expanded in any of the three conditions. The significantly strongest suppression was observed with cells expanded in FBS conditions, whereas higher ASC numbers were required to display suppression in HS or XF/SF conditions. In addition, statistically significant differences in protein secretion were observed between direct versus indirect cocultures and between different culture conditions. The characteristic immunophenotype of ASCs was maintained in all conditions. However, in XF/SF conditions, a significantly lower expression of CD54 (intercellular adhesion molecule 1) and a higher expression of CD45 (lymphocyte common antigen) was observed at a low passage number. Although culture conditions have an effect on the immunogenicity, immunosuppression, and protein secretion profile of ASCs, our findings demonstrated that ASCs have low immunogenicity and promising immunosuppressive potential whether cultured in FBS, HS, or XF

  11. Changes in levels of cell wall constituents in wheat seedlings grown under continuous hypergravity conditions

    NASA Astrophysics Data System (ADS)

    Wakabayashi, K.; Soga, K.; Kamisaka, S.; Hoson, T.

    Effects of continuous hypergravity stimuli on the amounts and composition of cell wall constituents were investigated in wheat shoots. Hypergravity (300 g) treatment for three days after germination increased the net amount of cell wall polysaccharides such as hemicellulose and cellulose, but reduced the shoot elongation. As a result, the amount of cell wall polysaccharides per unit length of shoot increased under hypergravity. The hemicellulose fraction contained polysaccharides in the middle and low molecular mass range (5 kDa-1 MDa) and increased in response to hypergravity. Also, the amounts of arabinose (Ara) and xylose (Xyl), the major sugar components of the hemicellulose fraction, increased under hypergravity conditions. In addition to wall polysaccharides, hypergravity increased the amounts of cell wall-bound phenolic acids, such as ferulic acid (FA) and diferulic acid (DFA). Furthermore, the activity of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) was enhanced under hypergravity conditions. These results suggest that continuous hypergravity stimulates the synthesis of cell wall constituents, especially hemicellulosic arabinoxylans and cell wall-bound FA and DFA in wheat shoots. The increased PAL activity may promote the formation of FA and DFA. These changes in cell wall architecture may be involved in making rigid and tough cell walls under hypergravity conditions and thereby contribute to the ability of plant to sustain their structures against gravitational stimuli.

  12. Low calcium culture condition induces mesenchymal cell-like phenotype in normal human epidermal keratinocytes

    SciTech Connect

    Takagi, Ryo; Yamato, Masayuki; Murakami, Daisuke; Sugiyama, Hiroaki; Okano, Teruo

    2011-08-26

    Highlights: {yields} Normal human epidermal keratinocytes serially cultured under low calcium concentration were cytokeratin and vimentin double positive cells. {yields} The human keratinocytes expressed some epithelial stem/progenitor cell makers, mesenchymal cell markers, and markers of epithelial-mesenchymal transition. {yields} Mesenchymal cell-like phenotype in the keratinocytes was suppressed under high-calcium condition. -- Abstract: Epithelial-mesenchymal transition (EMT) is an important cellular phenomenon in organ developments, cancer invasions, and wound healing, and many types of transformed cell lines are used for investigating for molecular mechanisms of EMT. However, there are few reports for EMT in normal human epithelial cells, which are non-transformed or non-immortalized cells, in vitro. Therefore, normal human epidermal keratinocytes (NHEK) serially cultured in low-calcium concentration medium (LCM) were used for investigating relations between differentiation and proliferation and mesenchymal-like phenotype in the present study, since long-term cultivation of NHEK is achieved in LCM. Interestingly, NHEK serially cultured in LCM consisted essentially of cytokeratin-vimentin double positive cells (98%), although the NHEK exhibited differentiation under high-calcium culture condition with 3T3 feeder layer. The vimentin expression was suppressed under high-calcium condition. These results may indicate the importance of mesenchymal-like phenotype for serially cultivation of NHEK in vitro.

  13. Development and validation of characteristic boundary conditions for cell-centered Euler flow calculations

    NASA Astrophysics Data System (ADS)

    van den Berg, J. I.; Boerstoel, J. W.

    An overview of the development, analysis, and numerical validation of a solid-wall boundary condition for cell-centered Euler-flow calculations is presented. This solid-wall boundary condition is provided by the theory of characteristics, and is based on a central-difference scheme. The boundary condition was developed to investigate the effect of various boundary-condition algorithms on the accuracy of calculation results for three-dimensional Euler flows around delta wings. A mathematical analysis of the boundary condition was performed. The numerical validation consists of a comparison of calculation results with various boundary conditions. Also discretization and convergence errors were investigated. As a test case, the NLR 7301 profile under supercritical, shock-free flow conditions of M = 0.721, alpha = -0.194 deg, were chosen.

  14. Systems biology. Conditional density-based analysis of T cell signaling in single-cell data.

    PubMed

    Krishnaswamy, Smita; Spitzer, Matthew H; Mingueneau, Michael; Bendall, Sean C; Litvin, Oren; Stone, Erica; Pe'er, Dana; Nolan, Garry P

    2014-11-28

    Cellular circuits sense the environment, process signals, and compute decisions using networks of interacting proteins. To model such a system, the abundance of each activated protein species can be described as a stochastic function of the abundance of other proteins. High-dimensional single-cell technologies, such as mass cytometry, offer an opportunity to characterize signaling circuit-wide. However, the challenge of developing and applying computational approaches to interpret such complex data remains. Here, we developed computational methods, based on established statistical concepts, to characterize signaling network relationships by quantifying the strengths of network edges and deriving signaling response functions. In comparing signaling between naïve and antigen-exposed CD4(+) T lymphocytes, we find that although these two cell subtypes had similarly wired networks, naïve cells transmitted more information along a key signaling cascade than did antigen-exposed cells. We validated our characterization on mice lacking the extracellular-regulated mitogen-activated protein kinase (MAPK) ERK2, which showed stronger influence of pERK on pS6 (phosphorylated-ribosomal protein S6), in naïve cells as compared with antigen-exposed cells, as predicted. We demonstrate that by using cell-to-cell variation inherent in single-cell data, we can derive response functions underlying molecular circuits and drive the understanding of how cells process signals.

  15. Simulated conditions of microgravity suppress progesterone production by luteal cells of the pregnant rat

    NASA Technical Reports Server (NTRS)

    Bhat, G. K.; Yang, H.; Sridaran, R.

    2001-01-01

    The purpose of this study was to assess whether simulated conditions of microgravity induce changes in the production of progesterone by luteal cells of the pregnant rat ovary using an in vitro model system. The microgravity environment was simulated using either a high aspect ratio vessel (HARV) bioreactor with free fall or a clinostat without free fall of cells. A mixed population of luteal cells isolated from the corpora lutea of day 8 pregnant rats was attached to cytodex microcarrier beads (cytodex 3). These anchorage dependent cells were placed in equal numbers in the HARV or a spinner flask control vessel in culture conditions. It was found that HARV significantly reduced the daily production of progesterone from day 1 through day 8 compared to controls. Scanning electron microscopy showed that cells attached to the microcarrier beads throughout the duration of the experiment in both types of culture vessels. Cells cultured in chamber slide flasks and placed in a clinostat yielded similar results when compared to those in the HARV. Also, when they were stained by Oil Red-O for lipid droplets, the clinostat flasks showed a larger number of stained cells compared to control flasks at 48 h. Further, the relative amount of Oil Red-O staining per milligram of protein was found to be higher in the clinostat than in the control cells at 48 h. It is speculated that the increase in the level of lipid content in cells subjected to simulated conditions of microgravity may be due to a disruption in cholesterol transport and/or lesions in the steroidogenic pathway leading to a fall in the synthesis of progesterone. Additionally, the fall in progesterone in simulated conditions of microgravity could be due to apoptosis of luteal cells.

  16. Conditioning causes an increase in Glucose Transporter-4 levels in mononuclear cells in sled dogs

    PubMed Central

    Schnurr, Theresia M.; Reynolds, Arleigh J.; Gustafson, Sally J.; Duffy, Lawrence K.; Dunlap, Kriya L.

    2014-01-01

    This study was designed to investigate the effects of physical conditioning on the expression of the insulin sensitive glucose transporter 4 protein (GLUT4) on mononuclear cells and HOMA-IR levels in dogs and compared to results reported in human skeletal muscle and the skeletal muscle of rodent models. Blood was sampled from conditioned dogs (n=8) and sedentary dogs (n=8). The conditioned dogs were exercised four months prior the experiment and were following a uniform training protocol, whereas the sedentary dogs were not. GLUT4 expression in mononuclear cells and plasma insulin levels were measured using commercially available enzyme-linked immunosorbent assay (ELISA). Blood glucose levels were determined using blood plasma. HOMA-IR was calculated using plasma insulin and blood glucose levels using the linear approximation formula. Our results indicate that the state of conditioning had a significant effect on the GLUT4 expression at the surface of mononuclear cells. HOMA-IR was also affected by conditioning in dogs. GLUT4 levels in mononuclear cells of sled dogs were inversely correlated with the homeostasis model assessment of insulin sensitivity. This study demonstrates that conditioning increases GLUT4 levels in mononuclear cells of sled dogs as it has been previously reported in skeletal muscle. Our results support the potential of white blood cells as a proxy tissue for studying insulin signaling and may lead to development of a minimally invasive and direct marker of insulin resistance. This may be the first report of GLUT4 in mononuclear cells in response to exercise and measured with ELISA. PMID:25236492

  17. Principles and design of a novel magnetic force mechanical conditioning bioreactor for tissue engineering, stem cell conditioning, and dynamic in vitro screening.

    PubMed

    Dobson, Jon; Cartmell, Sarah H; Keramane, Ahmed; El Haj, Alicia J

    2006-09-01

    Mechanical conditioning of cells and tissue constructs in bioreactors is an important factor in determining the properties of tissue being produced. Mechanical conditioning within a bioreactor environment, however, has proven difficult. This paper presents the theoretical basis, design, and initial results of a mechanical conditioning system for cell and tissue culture which is based on biocompatible magnetic micro- and nanoparticles acting as a remote stress mechanism without invasion of the sterile bioreactor environment.

  18. Chromosomal variability of human mesenchymal stem cells cultured under hypoxic conditions

    PubMed Central

    Ueyama, Hanae; Horibe, Tomohisa; Hinotsu, Shiro; Tanaka, Tomoaki; Inoue, Takeomi; Urushihara, Hisashi; Kitagawa, Akira; Kawakami, Koji

    2012-01-01

    Abstract Bone marrow derived human mesenchymal stem cells (hMSCs) have attracted great interest from both bench and clinical researchers because of their pluripotency and ease of expansion ex vivo. However, these cells do finally reach a senescent stage and lose their multipotent potential. Proliferation of these cells is limited up to the time of their senescence, which limits their supply, and they may accumulate chromosomal changes through ex vivo culturing. The safe, rapid expansion of hMSCs is critical for their clinical application. Chromosomal aberration is known as one of the hallmarks of human cancer, and therefore it is important to understand the chromosomal stability and variability of ex vivo expanded hMSCs before they are used widely in clinical applications. In this study, we examined the effects of culturing under ambient (20%) or physiologic (5%) O2 concentrations on the rate of cell proliferation and on the spontaneous transformation of hMSCs in primary culture and after expansion, because it has been reported that culturing under hypoxic conditions accelerates the propagation of hMSCs. Bone marrow samples were collected from 40 patients involved in clinical research. We found that hypoxic conditions promote cell proliferation more favourably than normoxic conditions. Chromosomal aberrations, including structural instability or aneuploidy, were detected in significantly earlier passages under hypoxic conditions than under normoxic culture conditions, suggesting that amplification of hMSCs in a low-oxygen environment facilitated chromosomal instability. Furthermore, smoothed hazard-function modelling of chromosomal aberrations showed increased hazard after the fourth passage under both sets of culture conditions, and showed a tendency to increase the detection rate of primary karyotypic abnormalities among donors aged 60 years and over. In conclusion, we propose that the continuous monitoring of hMSCs will be required before they are used in

  19. In situ microscopic observation of chitin and fungal cells with chitinous cell walls in hydrothermal conditions

    PubMed Central

    Deguchi, Shigeru; Tsujii, Kaoru; Horikoshi, Koki

    2015-01-01

    Recent findings of intact chitin in fossil records suggest surprisingly high recalcitrance of this biopolymer during hydrothermal treatments. We also know in the experience of everyday life that mushroom, cells of which have chitinous cell walls, do not fall apart however long they are simmered. We used in situ optical microscopy to examine chitin and fungal cells with chitinous cell walls during hydrothermal treatments, and obtained direct evidence that they remained undegraded at temperatures well over 200 °C. The results show very hot and compressed water is needed to make mushrooms mushy. PMID:26148792

  20. Analysis of nucleocytoplasmic transport in digitonin-permeabilized cells under different cellular conditions.

    PubMed

    Furuta, Maiko; Kose, Shingo; Kehlenbach, Ralph H; Imamoto, Naoko

    2014-01-01

    The regulation of nucleocytoplasmic transport is crucial not only for basic cellular activities but also for physiological adaptation to specific situation during the cell cycle, development, or stress. Although a wide variety of transport pathways have been identified in eukaryotic cells, the functional significance of their multiplicity remains unclear. The best-characterized nuclear transport receptors (NTRs) are the members of the importin β family (karyopherin, transportin) whose association with specific cargoes is regulated by the GTPase Ran. In this chapter, we first provide an overview of the various expression vectors used to purify recombinant NTRs. We then describe two sets of recent examples of using well-established digitonin-permeabilized cell-free transport systems in mammalian cells to mimic different cellular conditions in living cells: normal/heat-shock conditions and interphase/mitosis. In the former case, physiological regulation impacts different transport pathways in opposite ways. In the latter case, the importin β-Ran system is used at different cell-cycle stages but with the same biochemical principle to specify the nuclear localization and chromatin loading of a specific protein, respectively. This in vitro transport assay, when adapted to specific cellular conditions or particular substrates, should help to uncover specific transport pathways or transport factors function under different cellular conditions.

  1. From microgravity to osmotic conditions: mechanical integration of plant cells in response to stress

    NASA Astrophysics Data System (ADS)

    Wojtaszek, Przemyslaw; Kasprowicz, Anna; Michalak, Michal; Janczara, Renata; Volkmann, Dieter; Baluska, Frantisek

    Chemical reactions and interactions between molecules are commonly thought of as being at the basis of Life. Research of recent years, however, is more and more evidently indicating that physical forces are profoundly affecting the functioning of life at all levels of its organiza-tion. To detect and to respond to such forces, plant cells need to be integrated mechanically. Cell walls are the outermost functional zone of plant cells. They surround the individual cells, and also form a part of the apoplast. In cell suspensions, cell walls are embedded in the cul-ture medium which can be considered as a superapoplast. Through physical and chemical interactions they provide a basis for the structural and functional cell wall-plasma membrane-cytoskeleton (WMC) continuum spanning the whole cell. Here, the working of WMC contin-uum, and the participation of signalling molecules, like NO, would be presented in the context of plant responses to stress. In addition, the effects of the changing composition of WMC continuum will be considered, with particular attention paid to the modifications of the WMC components. Plant cells are normally adapted to changing osmotic conditions, resulting from variable wa-ter availability. The appearance of the osmotic stress activates adaptory mechanisms. If the strength of osmotic stress grows relatively slowly over longer period of time, the cells are able to adapt to conditions that are lethal to non-adapted cells. During stepwise adaptation of tobacco BY-2 suspension cells to the presence of various osmotically active agents, cells diverged into independent, osmoticum type-specific lines. In response to ionic agents (NaCl, KCl), the adhe-sive properties were increased and randomly dividing cells formed clumps, while cells adapted to nonionic osmotica (mannitol, sorbitol, PEG) revealed ordered pattern of precisely positioned cell divisions, resulting in the formation of long cell files. Changes in the growth patterns were accompanied by

  2. Conditioned medium from alternatively activated macrophages induce mesangial cell apoptosis via the effect of Fas

    SciTech Connect

    Huang, Yuan; Luo, Fangjun; Li, Hui; Jiang, Tao; Zhang, Nong

    2013-11-15

    During inflammation in the glomerulus, the proliferation of myofiroblast-like mesangial cells is commonly associated with the pathological process. Macrophages play an important role in regulating the growth of resident mesangial cells in the glomeruli. Alternatively activated macrophage (M2 macrophage) is a subset of macrophages induced by IL-13/IL-4, which is shown to play a repair role in glomerulonephritis. Prompted by studies of development, we performed bone marrow derived macrophage and rat mesangial cell co-culture study. Conditioned medium from IL-4 primed M2 macrophages induced rat mesangial cell apoptosis. The pro-apoptotic effect of M2 macrophages was demonstrated by condensed nuclei stained with Hoechst 33258, increased apoptosis rates by flow cytometry analysis and enhanced caspase-3 activation by western blot. Fas protein was up-regulated in rat mesangial cells, and its neutralizing antibody ZB4 partly inhibited M2 macrophage-induced apoptosis. The up-regulated arginase-1 expression in M2 macrophage also contributed to this apoptotic effect. These results indicated that the process of apoptosis triggered by conditioned medium from M2 macrophages, at least is partly conducted through Fas in rat mesangial cells. Our findings provide compelling evidence that M2 macrophages control the growth of mesangial cells in renal inflammatory conditions. - Highlights: • Conditioned-medium from M2 macrophages induces rat mesangial cell (MsC) apoptosis. • M2 macrophage conditioned medium exerts its pro-apoptotic effects via Fas ligand. • Arginase-1 activity in M2 macrophages plays a role in inducing apoptosis in rat MsC.

  3. Morphogenesis of human embryonic stem cells into mature neurons under in vitro culture conditions

    PubMed Central

    Shroff, Geeta

    2016-01-01

    AIM To describe the morphogenesis of different neuronal cells from the human embryonic stem cell (hESC) line, SCT-N, under in vitro culture conditions. METHODS The directed neuronal cell line was produced from a single, spare, pre-implantation stage fertilized ovum that was obtained during a natural in vitro fertilization process. The hESCs were cultured and maintained as per our proprietary in-house technology in a Good Manufacturing Practice, Good Laboratory Practice and Good Tissue Practice compliant laboratory. The cell line was derived and incubated in aerobic conditions. The cells were examined daily under a phase contrast microscope for their growth and differentiation. RESULTS Different neural progenitor cells (NPCs) and differentiating neurons were observed under the culture conditions. Multipotent NPCs differentiated into all three types of nervous system cells, i.e., neurons, oligodendrocytes and astrocytes. Small projections resembling neurites or dendrites, and protrusion coming out of the cells, were observed. Differentiating cells were observed at day 18 to 20. The differentiating neurons, neuronal bodies, axons, and neuronal tissue were observed on day 21 and day 30 of the culture. On day 25 and day 30, prominent neurons, axons and neuronal tissue were observed under phase contrast microscopy. 4’, 6-diamidino-2-phenylindole staining also indicated the pattern of differentiating neurons, axonal structure and neuronal tissue. CONCLUSION This study describes the generation of different neuronal cells from an hESC line derived from biopsy of blastomeres at the two-cell cleavage stage from a discarded embryo. PMID:27909687

  4. T-Cell Activation under Hypoxic Conditions Enhances IFN-γ Secretion

    PubMed Central

    Roman, Jessica; Rangasamy, Tirumalai; Guo, Jia; Sugunan, Siva; Meednu, Nida; Packirisamy, Gopinath; Shimoda, Larissa A.; Golding, Amit; Semenza, Gregg; Georas, Steve N.

    2010-01-01

    Secondary lymphoid organs and peripheral tissues are characterized by hypoxic microenvironments, both in the steady state and during inflammation. Although hypoxia regulates T-cell metabolism and survival, very little is known about whether or how hypoxia influences T-cell activation. We stimulated mouse CD4+ T cells in vitro with antibodies directed against the T-cell receptor (CD3) and CD28 under normoxic (20% O2) and hypoxic (1% O2) conditions. Here we report that stimulation under hypoxic conditions augments the secretion of effector CD4+ T-cell cytokines, especially IFN-γ. The enhancing effects of hypoxia on IFN-γ secretion were independent of mouse strain, and were also unaffected using CD4+ T cells from mice lacking one copy of the gene encoding hypoxia-inducible factor-1α. Using T cells from IFN-γ receptor–deficient mice and promoter reporter studies in transiently transfected Jurkat T cells, we found that the enhancing effects of hypoxia on IFN-γ expression were not due to effects on IFN-γ consumption or proximal promoter activity. In contrast, deletion of the transcription factor, nuclear erythroid 2 p45–related factor 2 attenuated the enhancing effect of hypoxia on IFN-γ secretion and other cytokines. We conclude that hypoxia is a previously underappreciated modulator of effector cytokine secretion in CD4+ T cells. PMID:19372249

  5. Lysophosphatidic acid enhances survival of human CD34+ cells in ischemic conditions

    PubMed Central

    Kostic, Ivana; Fidalgo-Carvalho, Isabel; Aday, Sezin; Vazão, Helena; Carvalheiro, Tiago; Grãos, Mário; Duarte, António; Cardoso, Carla; Gonçalves, Lino; Carvalho, Lina; Paiva, Artur; Ferreira, Lino

    2015-01-01

    Several clinical trials are exploring therapeutic effect of human CD34+ cells in ischemic diseases, including myocardial infarction. Unfortunately, most of the cells die few days after delivery. Herein we show that lysophosphatidic acid (LPA)-treated human umbilical cord blood-derived CD34+ cells cultured under hypoxic and serum-deprived conditions present 2.2-fold and 1.3-fold higher survival relatively to non-treated cells and prostaglandin E2-treated cells, respectively. The pro-survival effect of LPA is concentration- and time-dependent and it is mediated by the activation of peroxisome proliferator-activator receptor γ (PPARγ) and downstream, by the activation of pro-survival ERK and Akt signaling pathways and the inhibition of mitochondrial apoptotic pathway. In hypoxia and serum-deprived culture conditions, LPA induces CD34+ cell proliferation without maintaining the their undifferentiating state, and enhances IL-8, IL-6 and G-CSF secretion during the first 12 h compared to non-treated cells. LPA-treated CD34+ cells delivered in fibrin gels have enhanced survival and improved cardiac fractional shortening at 2 weeks on rat infarcted hearts as compared to hearts treated with placebo. We have developed a new platform to enhance the survival of CD34+ cells using a natural and cost-effective ligand and demonstrated its utility in the preservation of the functionality of the heart after infarction. PMID:26553339

  6. Regulation of osteogenic differentiation of human adipose-derived stem cells by controlling electromagnetic field conditions

    PubMed Central

    Kang, Kyung Shin; Hong, Jung Min; Kang, Jo A; Rhie, Jong-Won; Jeong, Young Hun; Cho, Dong-Woo

    2013-01-01

    Many studies have reported that an electromagnetic field can promote osteogenic differentiation of mesenchymal stem cells. However, experimental results have differed depending on the experimental and environmental conditions. Optimization of electromagnetic field conditions in a single, identified system can compensate for these differences. Here we demonstrated that specific electromagnetic field conditions (that is, frequency and magnetic flux density) significantly regulate osteogenic differentiation of adipose-derived stem cells (ASCs) in vitro. Before inducing osteogenic differentiation, we determined ASC stemness and confirmed that the electromagnetic field was uniform at the solenoid coil center. Then, we selected positive (30/45 Hz, 1 mT) and negative (7.5 Hz, 1 mT) osteogenic differentiation conditions by quantifying alkaline phosphate (ALP) mRNA expression. Osteogenic marker (for example, runt-related transcription factor 2) expression was higher in the 30/45 Hz condition and lower in the 7.5 Hz condition as compared with the nonstimulated group. Both positive and negative regulation of ALP activity and mineralized nodule formation supported these responses. Our data indicate that the effects of the electromagnetic fields on osteogenic differentiation differ depending on the electromagnetic field conditions. This study provides a framework for future work on controlling stem cell differentiation. PMID:23306704

  7. Ebselen Preserves Tissue-Engineered Cell Sheets and their Stem Cells in Hypothermic Conditions

    PubMed Central

    Katori, Ryosuke; Hayashi, Ryuhei; Kobayashi, Yuki; Kobayashi, Eiji; Nishida, Kohji

    2016-01-01

    Clinical trials have been performed using autologous tissue-engineered epithelial cell sheets for corneal regenerative medicine. To improve stem cell-based therapy for convenient clinical practice, new techniques are required for preserving reconstructed tissues and their stem/progenitor cells until they are ready for use. In the present study, we screened potential preservative agents and developed a novel medium for preserving the cell sheets and their stem/progenitor cells; the effects were evaluated with a luciferase-based viability assay. Nrf2 activators, specifically ebselen, could maintain high ATP levels during preservation. Ebselen also showed a strong influence on maintenance of the viability, morphology, and stem cell function of the cell sheets preserved under hypothermia by protecting them from reactive oxygen species-induced damage. Furthermore, ebselen drastically improved the preservation performance of human cornea tissues and their stem cells. Therefore, ebselen shows good potential as a useful preservation agent in regenerative medicine as well as in cornea transplantation. PMID:27966584

  8. Glial expression of Swiss cheese (SWS), the Drosophila orthologue of neuropathy target esterase (NTE), is required for neuronal ensheathment and function

    PubMed Central

    Dutta, Sudeshna; Rieche, Franziska; Eckl, Nina; Duch, Carsten; Kretzschmar, Doris

    2016-01-01

    ABSTRACT Mutations in Drosophila Swiss cheese (SWS) or its vertebrate orthologue neuropathy target esterase (NTE), respectively, cause progressive neuronal degeneration in Drosophila and mice and a complex syndrome in humans that includes mental retardation, spastic paraplegia and blindness. SWS and NTE are widely expressed in neurons but can also be found in glia; however, their function in glia has, until now, remained unknown. We have used a knockdown approach to specifically address SWS function in glia and to probe for resulting neuronal dysfunctions. This revealed that loss of SWS in pseudocartridge glia causes the formation of multi-layered glial whorls in the lamina cortex, the first optic neuropil. This phenotype was rescued by the expression of SWS or NTE, suggesting that the glial function is conserved in the vertebrate protein. SWS was also found to be required for the glial wrapping of neurons by ensheathing glia, and its loss in glia caused axonal damage. We also detected severe locomotion deficits in glial sws-knockdown flies, which occurred as early as 2 days after eclosion and increased further with age. Utilizing the giant fibre system to test for underlying functional neuronal defects showed that the response latency to a stimulus was unchanged in knockdown flies compared to controls, but the reliability with which the neurons responded to increasing frequencies was reduced. This shows that the loss of SWS in glia impairs neuronal function, strongly suggesting that the loss of glial SWS plays an important role in the phenotypes observed in the sws mutant. It is therefore likely that changes in glia also contribute to the pathology observed in humans that carry mutations in NTE. PMID:26634819

  9. Optimal culture conditions for the generation of natural killer cell-induced dendritic cells for cancer immunotherapy.

    PubMed

    Nguyen-Pham, Thanh-Nhan; Yang, Deok-Hwan; Nguyen, Truc-Anh Thi; Lim, Mi-Seon; Hong, Cheol Yi; Kim, Mi-Hyun; Lee, Hyun Ju; Lee, Youn-Kyung; Cho, Duck; Bae, Soo-Young; Ahn, Jae-Sook; Kim, Yeo-Kyeoung; Chung, Ik-Joo; Kim, Hyeoung-Joon; Lee, Je-Jung

    2012-01-01

    Dendritic cell (DC)-based vaccines continue to be considered an attractive tool for cancer immunotherapy. DCs require an additional signal from the environment or other immune cells to polarize the development of immune responses toward T helper 1 (Th1) or Th2 responses. DCs play a role in natural killer (NK) cell activation, and NK cells are also able to activate and induce the maturation of DCs. We investigated the types of NK cells that can induce the maturation and enhanced function of DCs and the conditions under which these interactions occur. DCs that were activated by resting NK cells in the presence of inflammatory cytokines exhibited increased expression of several costimulatory molecules and an enhanced ability to produce IL-12p70. NK cell-stimulated DCs potently induced Th1 polarization and exhibited the ability to generate tumor antigen-specific cytotoxic T lymphocyte responses. Our data demonstrate that functional DCs can be generated by coculturing immature DCs with freshly isolated resting NK cells in the presence of Toll-like receptor agonists and proinflammatory cytokines and that the resulting DCs effectively present antigens to induce tumor-specific T-cell responses, which suggests that these cells may be useful for cancer immunotherapy.

  10. Performance enhancement of a direct borohydride fuel cell in practical running conditions

    NASA Astrophysics Data System (ADS)

    Kim, Cheolhwan; Kim, Kyu-Jung; Ha, Man Yeong

    To investigate the possibility of a cost-effective direct borohydride fuel cell (DBFC), the performance enhancement of a single cell is investigated under practical running conditions by adopting non-precious metal for the anode. Fluorinated Zr-based AB 2-type hydrogen storage alloy with an effective area of 100 cm 2 is selected as the anode catalyst. To minimize pressure loss from the enlarged cell size, a parallel-type anode channel is designed, then the principal reasons for performance degradation are analyzed. Single-cell performance is mainly enhanced by adopting a corrugated anode design, applying an anti-corrosion coating on the cathode channel, and controlling the fuel flow-rate and air humidity. The cell performance is estimated simply by measuring the wall temperature of the cell.

  11. Mineralization of bone-related SaOS-2 cells under physiological hypoxic conditions.

    PubMed

    Müller, Werner E G; Schröder, Heinz C; Tolba, Emad; Diehl-Seifert, Bärbel; Wang, Xiaohong

    2016-01-01

    Inorganic polyphosphate (polyP) is a physiological energy-rich polymer with multiple phosphoric anhydride bonds. In cells such as bone-forming osteoblasts, glycolysis is the main pathway generating metabolic energy in the form of ATP. In the present study, we show that, under hypoxic culture conditions, the growth/viability of osteoblast-like SaOS-2 cells is not impaired. The addition of polyP to those cells, administered as amorphous calcium polyP nanoparticles (aCa-polyP-NP; approximate size 100 nm), significantly increased the proliferation of the cells. In the presence of polyP, the cells produce significant levels of lactate, the end product of anaerobic glycolysis. Under those conditions, an eight-fold increase in the steady-state level of the membrane-associated carbonic anhydrase IX is found, as well as a six-fold induction of the hypoxia-inducible factor 1. Consequently, biomineral formation onto the SaOS-2 cells decreases under low oxygen tension. If the polyP nanoparticles are added to the cells, the degree of mineralization is enhanced. These changes had been measured also in human mesenchymal stem cells. The assumption that the bicarbonate, generated by the carbonic anhydrase in the presence of polyP under low oxygen, is deposited as a constituent of the bioseeds formed during initial hydroxyapatite formation is corroborated by the identification of carbon besides of calcium, oxygen and phosphorus in the initial biomineral deposit onto the cells using the sensitive technology of high-resolution energy dispersive spectrometry mapping. Based on these data, we conclude that polyP is required for the supply of metabolic energy during bone mineral formation under physiological, hypoxic conditions, acting as a 'metabolic fuel' for the cells to grow.

  12. Optimization of Cell Adhesion on Mg Based Implant Materials by Pre-Incubation under Cell Culture Conditions

    PubMed Central

    Willumeit, Regine; Möhring, Anneke; Feyerabend, Frank

    2014-01-01

    Magnesium based implants could revolutionize applications where orthopedic implants such as nails, screws or bone plates are used because they are load bearing and degrade over time. This prevents a second surgery to remove conventional implants. To improve the biocompatibility we studied here if and for how long a pre-incubation of the material under cell culture conditions is favorable for cell attachment and proliferation. For two materials, Mg and Mg10Gd1Nd, we could show that 6 h pre-incubation are already enough to form a natural protective layer suitable for cell culture. PMID:24857908

  13. Growth and cell wall changes in stem organs under microgravity and hypergravity conditions

    NASA Astrophysics Data System (ADS)

    Hoson, Takayuki; Soga, Kouichi; Wakabayashi, Kazuyuki; Kamisaka, Seiichiro

    Gravity strongly influences plant growth and development, which is fundamentally brought about by modifications to the properties of the cell wall. We have examined the changes in growth and cell wall properties in seedling organs under hypergravity conditions produced by centrifugation and under microgravity conditions in space. Hypergravity stimuli have been shown to decrease the growth rate of various seedling organs. When hypergravity suppressed elongation growth, a decrease in cell wall extensibility (an increase in cell wall rigidity) was induced. Hypergravity has also been shown to increase cell wall thickness in various mate-rials. In addition, a polymerization of certain matrix polysaccharides was brought about by hypergravity: in dicotyledons hypergravity increased the molecular size of xyloglucans, whereas hypergravity increased that of 1,3,1,4-β-glucans in monocotyledonous Gramineae. These mod-ifications to cell wall metabolism may be responsible for a decrease in cell wall extensibility, leading to growth suppression under hypergravity conditions. How then does microgravity in-fluence growth and cell wall properties? Here, there was a possibility that microgravity might induce changes similar to those by hypergravity, because plants have evolved and adapted to 1 g condition for more than 400 million years. However, the changes observed under microgravity conditions in space were just opposite to those induced by hypergravity: stimulation of elonga-tion growth, an increase in cell wall extensibility, and a decrease in cell wall thickness as well as depolymerization of cell wall polysaccharides were brought about in space. Furthermore, growth and cell wall properties varied in proportion to the logarithm of the magnitude of grav-ity in the range from microgravity to hypergravity, as shown in the dose-response relation in light and hormonal responses. Thus, microgravity may be a `stress-less' environment for plant seedlings to grow and develop

  14. Effectiveness of dye sensitised solar cell under low light condition using wide band dye

    SciTech Connect

    Sahmer, Ahmad Zahrin Mohamed, Norani Muti Zaine, Siti Nur Azella

    2015-07-22

    Dye sensistised solar cell (DSC) based on nanocrystalline TiO{sub 2} has the potential to be used in indoor consumer power application. In realizing this, the DSC must be optimized to generate power under low lighting condition and under wider visible light range. The use of wide band dye N749 which has a wider spectrum sensitivity increases the photon conversion to electron between the visible light spectrums of 390nm to 700nm. This paper reports the study on the effectiveness of the dye solar cell with N749 dye under low light condition in generating usable power which can be used for indoor consumer application. The DSC was fabricated using fluorine doped tin oxide (FTO) glass with screen printing method and the deposited TiO{sub 2} film was sintered at 500°C. The TiO{sub 2} coated FTO glass was then soaked in the N749 dye, assembled into test cell, and tested under the standard test condition at irradiance of 1000 W/m{sup 2} with AM1.5 solar soaker. The use of the 43T mesh for the dual pass screen printing TiO{sub 2} paste gives a uniform TiO{sub 2} film layer of 16 µm. The low light condition was simulated using 1/3 filtered irradiance with the solar soaker. The fabricated DSC test cell with the N749 dye was found to have a higher efficiency of 6.491% under low light condition compared to the N719 dye. Under the standard test condition at 1 sun the N749 test cell efficiency is 4.55%. The increases in efficiency is attributed to the wider spectral capture of photon of the DSC with N749 dye. Furthermore, the use of N749 dye is more effective under low light condition as the V{sub OC} decrement is less significant compared to the latter.

  15. Pavlovian Conditioning of Rat Mucosal Mast Cells to Secrete Rat Mast Cell Protease II

    NASA Astrophysics Data System (ADS)

    MacQueen, Glenda; Marshall, Jean; Perdue, Mary; Siegel, Shepard; Bienenstock, John

    1989-01-01

    Antigen (egg albumin) injections, which stimulate mucosal mast cells to secrete mediators, were paired with an audiovisual cue. After reexposure to the audiovisual cue, a mediator (rat mast cell protease II) was measured with a sensitive and specific assay. Animals reexposed to only the audiovisual cue released a quantity of protease not significantly different from animals reexposed to both the cue and the antigen; these groups released significantly more protease than animals that had received the cue and antigen in a noncontingent manner. The results support a role for the central nervous system as a functional effector of mast cell function in the allergic state.

  16. Amelioration of murine sickle cell disease by nonablative conditioning and γ-globin gene-corrected bone marrow cells.

    PubMed

    Pestina, Tamara I; Hargrove, Phillip W; Zhao, Huifen; Mead, Paul E; Smeltzer, Matthew P; Weiss, Mitchell J; Wilber, Andrew; Persons, Derek A

    2015-01-01

    Patients with severe sickle cell disease (SCD) are candidates for gene therapy using autologous hematopoietic stem cells (HSCs), but concomitant multi-organ disease may contraindicate pretransplant conditioning with full myeloablation. We tested whether nonmyeloablative conditioning, a regimen used successfully for allogeneic bone marrow transplantation of adult SCD patients, allows engraftment of γ-globin gene-corrected cells to a therapeutic level in the Berkeley mouse model of SCD. Animals transplanted according to this regimen averaged 35% engraftment of transduced hematopoietic stem cells with an average vector copy < 2.0. Fetal hemoglobin (HbF) levels ranged from 20 to 44% of total hemoglobin and approximately two-thirds of circulating red blood cells expressed HbF detected by immunofluorescence (F-cells). Gene therapy treatment of SCD mice ameliorated anemia, reduced hyperleukocytosis, improved renal function, and reduced iron accumulation in liver, spleen, and kidneys. Thus, modest levels of chimerism with donor cells expressing high levels of HbF from an insulated γ-globin lentiviral vector can improve the pathology of SCD in mice, thereby illustrating a potentially safe and effective strategy for gene therapy in humans.

  17. Amelioration of murine sickle cell disease by nonablative conditioning and γ-globin gene-corrected bone marrow cells

    PubMed Central

    Pestina, Tamara I; Hargrove, Phillip W; Zhao, Huifen; Mead, Paul E; Smeltzer, Matthew P; Weiss, Mitchell J; Wilber, Andrew; Persons, Derek A

    2015-01-01

    Patients with severe sickle cell disease (SCD) are candidates for gene therapy using autologous hematopoietic stem cells (HSCs), but concomitant multi-organ disease may contraindicate pretransplant conditioning with full myeloablation. We tested whether nonmyeloablative conditioning, a regimen used successfully for allogeneic bone marrow transplantation of adult SCD patients, allows engraftment of γ-globin gene-corrected cells to a therapeutic level in the Berkeley mouse model of SCD. Animals transplanted according to this regimen averaged 35% engraftment of transduced hematopoietic stem cells with an average vector copy < 2.0. Fetal hemoglobin (HbF) levels ranged from 20 to 44% of total hemoglobin and approximately two-thirds of circulating red blood cells expressed HbF detected by immunofluorescence (F-cells). Gene therapy treatment of SCD mice ameliorated anemia, reduced hyperleukocytosis, improved renal function, and reduced iron accumulation in liver, spleen, and kidneys. Thus, modest levels of chimerism with donor cells expressing high levels of HbF from an insulated γ-globin lentiviral vector can improve the pathology of SCD in mice, thereby illustrating a potentially safe and effective strategy for gene therapy in humans. PMID:26665131

  18. Cth2 Protein Mediates Early Adaptation of Yeast Cells to Oxidative Stress Conditions.

    PubMed

    Castells-Roca, Laia; Pijuan, Jordi; Ferrezuelo, Francisco; Bellí, Gemma; Herrero, Enrique

    2016-01-01

    Cth2 is an mRNA-binding protein that participates in remodeling yeast cell metabolism in iron starvation conditions by promoting decay of the targeted molecules, in order to avoid excess iron consumption. This study shows that in the absence of Cth2 immediate upregulation of expression of several of the iron regulon genes (involved in high affinity iron uptake and intracellular iron redistribution) upon oxidative stress by hydroperoxide is more intense than in wild type conditions where Cth2 is present. The oxidative stress provokes a temporary increase in the levels of Cth2 (itself a member of the iron regulon). In such conditions Cth2 molecules accumulate at P bodies-like structures when the constitutive mRNA decay machinery is compromised. In addition, a null Δcth2 mutant shows defects, in comparison to CTH2 wild type cells, in exit from α factor-induced arrest at the G1 stage of the cell cycle when hydroperoxide treatment is applied. The cell cycle defects are rescued in conditions that compromise uptake of external iron into the cytosol. The observations support a role of Cth2 in modulating expression of diverse iron regulon genes, excluding those specifically involved in the reductive branch of the high-affinity transport. This would result in immediate adaptation of the yeast cells to an oxidative stress, by controlling uptake of oxidant-promoting iron cations.

  19. Cth2 Protein Mediates Early Adaptation of Yeast Cells to Oxidative Stress Conditions

    PubMed Central

    Ferrezuelo, Francisco; Bellí, Gemma; Herrero, Enrique

    2016-01-01

    Cth2 is an mRNA-binding protein that participates in remodeling yeast cell metabolism in iron starvation conditions by promoting decay of the targeted molecules, in order to avoid excess iron consumption. This study shows that in the absence of Cth2 immediate upregulation of expression of several of the iron regulon genes (involved in high affinity iron uptake and intracellular iron redistribution) upon oxidative stress by hydroperoxide is more intense than in wild type conditions where Cth2 is present. The oxidative stress provokes a temporary increase in the levels of Cth2 (itself a member of the iron regulon). In such conditions Cth2 molecules accumulate at P bodies-like structures when the constitutive mRNA decay machinery is compromised. In addition, a null Δcth2 mutant shows defects, in comparison to CTH2 wild type cells, in exit from α factor-induced arrest at the G1 stage of the cell cycle when hydroperoxide treatment is applied. The cell cycle defects are rescued in conditions that compromise uptake of external iron into the cytosol. The observations support a role of Cth2 in modulating expression of diverse iron regulon genes, excluding those specifically involved in the reductive branch of the high-affinity transport. This would result in immediate adaptation of the yeast cells to an oxidative stress, by controlling uptake of oxidant-promoting iron cations. PMID:26824473

  20. Downregulation of Metabolic Activity Increases Cell Survival Under Hypoxic Conditions: Potential Applications for Tissue Engineering

    PubMed Central

    Kim, Jaehyun; Andersson, Karl-Erik; Jackson, John D.; Lee, Sang Jin; Atala, Anthony

    2014-01-01

    A major challenge to the success of cell-based implants for tissue regeneration is an insufficient supply of oxygen before host vasculature is integrated into the implants, resulting in premature cell death and dysfunction. Whereas increasing oxygenation to the implants has been a major focus in the field, our strategy is aimed at lowering oxygen consumption by downregulating cellular metabolism of cell-based implants. Adenosine, which is a purine nucleoside that functions as an energy transferring molecule, has been reported to increase under hypoxia, resulting in reducing the adenosine triphosphate (ATP) demands of the Na+/K+ ATPase. In the present study, we investigated whether adenosine could be used to downregulate cellular metabolism to achieve prolonged survival under hypoxic conditions. Murine myoblasts (C2C12) lacking a self-survival mechanism were treated with adenosine under 0.1% hypoxic stress. The cells, cultured in the presence of 5 mM adenosine, maintained their viability under hypoxia, and regained their normal growth and function of forming myotubes when transferred to normoxic conditions at day 11 without further supply of adenosine, whereas nontreated cells failed to survive. An increase in adenosine concentrations shortened the onset of reproliferation after transfer to normoxic conditions. This increase correlated with an increase in metabolic downregulation during the early phase of hypoxia. A higher intracellular ATP level was observed in adenosine-treated cells throughout the duration of hypoxia. This strategy of increasing cell survival under hypoxic conditions through downregulating cellular metabolism may be utilized for cell-based tissue regeneration applications as well as protecting tissues against hypoxic injuries. PMID:24524875

  1. Reduced Toxicity Conditioning and Allogeneic Hematopoietic Progenitor Cell Transplantation for Recessive Dystrophic Epidermolysis Bullosa.

    PubMed

    Geyer, Mark B; Radhakrishnan, Kavita; Giller, Roger; Umegaki, Noriko; Harel, Sivan; Kiuru, Maija; Morel, Kimberly D; LeBoeuf, Nicole; Kandel, Jessica; Bruckner, Anna; Fabricatore, Sandra; Chen, Mei; Woodley, David; McGrath, John; Baxter-Lowe, LeeAnn; Uitto, Jouni; Christiano, Angela M; Cairo, Mitchell S

    2015-09-01

    Recessive dystrophic epidermolysis bullosa is a severe, incurable, inherited blistering disease caused by COL7A1 mutations. Emerging evidence suggests hematopoietic progenitor cells (HPCs) can be reprogrammed into skin; HPC-derived cells can restore COL7 expression in COL7-deficient mice. We report two children with recessive dystrophic epidermolysis bullosa treated with reduced-toxicity conditioning and HLA-matched HPC transplantation.

  2. [Significant impact of different induction conditions on metabolic diversity of callus cell lines of Glycyrrhiza sp].

    PubMed

    Liu, Feng-Cai; Lv, Jian-Ming; Wu, Xiu-Zhen; Zhang, Wei

    2013-12-01

    The purpose of this study was to evaluate the impact of callus induction and culture conditions on secondary metabolic diversity of the callus cell lines of traditional Chinese medicinal plant Glycyrrhiza sp. (Glycyrrhiza) by combined chemical analysis and HPLC fingerprint. These callus induction conditions included two Glycyrrhiza species, two types of explants, light and dark conditions, and two combinations of hormones. The evaluation was firstly based on the contents of total flavonoids in the callus by chemical analysis and one way ANOVA. The content of total flavonoids in callus was significantly (P < 0.05) influenced by Glycyrrhiza species, light condition, and the combination of hormones. The callus was further evaluated using diversity factor based on the comparison of HPLC fingerprints of these callus cell lines. Diversity factor varies significantly for calli induced under different conditions, with the highest being at 0.45 under light condition and combination of hormones. These results provide important knowledge for the selection of suitable callus cell lines for the production of pharmacologically important secondary metabolites or bioactive fractions by in vitro culture of Glycyrrhiza sp.

  3. Hypoxia Pre-Conditioned Embryonic Mesenchymal Stem Cell Secretome Reduces IL-10 Production by Peripheral Blood Mononuclear Cells

    PubMed Central

    Lotfinia, Majid; Lak, Shirin; Ghahhari, Nastaran Mohammadi; Johari, Behrooz; Maghsood, Faezeh; Parsania, Sara; Tabrizi, Bahareh Sadegh; Kadivar, Mehdi

    2017-01-01

    Background: Mesenchymal stem cells (MSCs) are important candidates for MSC-based cellular therapy. Current paradigm states that MSCs support local progenitor cells in damaged tissue through paracrine signaling. Therefore, the study of paracrine effects and secretome of MSCs could lead to the appreciation of mechanisms and molecules associated with the therapeutic effects of these cells. This study analyzed anti-inflammatory and immune-modulatory effects of MSC secretomes derived from embryonic stem cells (ESCs) and bone marrow cells after hypoxia and normoxia preconditioning. Methods: ESCs differentiated into MSCs and characterized by flow cytometry as well as by differentiation into adipocytes and osteoblasts. The experimental groups were consisted of individual groups of ESC-MSCs and BM-MSCs (bone marrow-derived mesenchymal stromal cells), which were preconditioned with either hypoxia or normoxia for 24, 48 and 72 h. After collecting the cell-free medium from each treatment, secretomes were concentrated by centrifugal filters. Using a peripheral blood mononuclear cell (PBMC) assay and ELISA, IL-10 concentration in PBMCs was evaluated after their incubation with different secretomes from preconditioned and non-preconditioned MSCs. Results: A significant difference was observed between ESC-MSC normoxia and ESC-MSC hypoxia in IL-10 concentration, and normoxia secretomes increased IL-10 secretion from PBMCs. Moreover, the strongest IL-10 secretion from PBMCs could be detected after the stimulation by ESC-MSC conditioned secretomes, but not BM-MSC conditioned medium. Conclusions: Human hypoxia preconditioned ESC-MSC secretome indicated stronger immune-modulatory effects compared to BM-MSC conditioned medium. It could be suggested that induced MSCs confer less immune-modulatory effects but produce more inflammatory molecules such as tumor necrosis factor α, which needs further investigation. PMID:27132108

  4. Laminin-adherent versus suspension-non-adherent cell culture conditions for the isolation of cancer stem cells in the DAOY medulloblastoma cell line.

    PubMed

    de la Rosa, Javier; Sáenz Antoñanzas, Ander; Shahi, Mehdi H; Meléndez, Bárbara; Rey, Juan A; Castresana, Javier S

    2016-09-01

    Medulloblastoma (MB) is a highly malignant tumor of childhood. MB seems to be initiated and maintained by a small group of cells, known as cancer stem cells (CSCs). The CSC hypothesis suggests that a subset of tumor cells is able to proliferate, sustain the tumor, and develop chemoresistance, all of which make of CSC an interesting target for new anticancer therapies. The MB cell line DAOY was cultured in suspension by a medullosphere traditional culturing method and in adherent conditions by laminin-pre-coated flasks and serum-free medium enriched with specific growth factors. An increase in the stem features was shown when cells were successively cultured in hypoxia conditions. By contrast, a reduction in these properties was appreciated when cells were exposed to differentiation conditions. In addition, the CD133+ and CD133- subpopulations were isolated from cells grown in laminin-pre-coated flasks, and in vitro experiments showed that the CD133+ fraction represented the stem population and it could have CSC with a higher probability than the CD133- fraction. We can conclude that the laminin culture method in adherent conditions and the medullosphere traditional culturing method in suspension are similarly good for obtaining stem-like cells in the DAOY cell line.

  5. Human Umbilical Cord Wharton's Jelly Stem Cell Conditioned Medium Induces Tumoricidal Effects on Lymphoma Cells Through Hydrogen Peroxide Mediation.

    PubMed

    Lin, Hao Daniel; Fong, Chui-Yee; Biswas, Arijit; Choolani, Mahesh; Bongso, Ariff

    2016-09-01

    Several groups have reported that human umbilical cord Wharton's jelly stem cells (hWJSCs) possess unique tumoricidal properties against many cancers. However, the exact mechanisms as to how hWJSCs inhibit tumor growth are not known. Recent evidence suggests that exposure of cancer cells to high hydrogen peroxide (H2 O2 ) levels from H2 O2 -releasing drugs causes their death. We therefore explored whether the tumoricidal effect of hWJSCs on lymphoma cells was mediated via H2 O2 . We first exposed lymphoma cells to six different molecular weight cut-off (MWCO) concentrates of hWJSC-conditioned medium (hWJSC-CM) (3, 5, 10, 30, 50, 100 kDa) for 48 h. Since, the 3 kDa-MWCO concentrate showed the greatest cell inhibition we then investigated whether the tumoricidal effect of the specific 3 kDa-MWCO concentrate on two different lymphoma cell lines (Ramos and Toledo) was mediated via accumulation of H2 O2 . We used a battery of assays (MTT, propidium iodide, mitochondria membrane potential, apoptosis, cell cycle, oxidative stress enzymes, hydrogen peroxide, mitochondrial superoxide, hydroxyl radical, peroxynitrile anion, and lipid peroxidation) to test this mechanism. The hWJSC-CM-3 kDa MWCO concentrate significantly decreased cell viability and mitochondrial membrane potential and increased cell death and apoptosis in both lymphoma cell lines. There were significant increases in superoxide dismutase with concomitant decreases in glutathione peroxidase, catalase, and thioredoxin peroxidase activities. H2 O2 levels, mitochondrial superoxide, hydroxyl radical, peroxynitrile anion, and lipid peroxidation were also significantly increased in both lymphoma cell lines. The results suggested that the hWJSC-CM-3 kDa MWCO concentrate regulates cellular H2 O2 leading to a tumoricidal effect and may thus be a promising anti-lymphoma agent. J. Cell. Biochem. 117: 2045-2055, 2016. © 2016 Wiley Periodicals, Inc.

  6. Peripheral self-reactivity regulates antigen-specific CD8 T-cell responses and cell division under physiological conditions

    PubMed Central

    Swee, Lee Kim; Tan, Zhen Wei; Sanecka, Anna; Yoshida, Nagisa; Patel, Harshil; Grotenbreg, Gijsbert; Ploegh, Hidde L.

    2016-01-01

    T-cell identity is established by the expression of a clonotypic T-cell receptor (TCR), generated by somatic rearrangement of TCRα and β genes. The properties of the TCR determine both the degree of self-reactivity and the repertoire of antigens that can be recognized. For CD8 T cells, the relationship between TCR identity—hence reactivity to self—and effector function(s) remains to be fully understood and has rarely been explored outside of the H-2b haplotype. We measured the affinity of three structurally distinct CD8 T-cell-derived TCRs that recognize the identical H-2 Ld-restricted epitope, derived from the Rop7 protein of Toxoplasma gondii. We used CD8 T cells obtained from mice generated by somatic cell nuclear transfer as the closest approximation of primary T cells with physiological TCR rearrangements and TCR expression levels. First, we demonstrate the common occurrence of secondary rearrangements in endogenously rearranged loci. Furthermore, we characterized and compared the response of Rop7-specific CD8 T-cell clones upon Toxoplasma gondii infection as well as effector function and TCR signalling upon antigenic stimulation in vitro. Antigen-independent TCR cross-linking in vitro uncovered profound intrinsic differences in the effector functions between T-cell clones. Finally, by assessing the degree of self-reactivity and comparing the transcriptomes of naive Rop7 CD8 T cells, we show that lower self-reactivity correlates with lower effector capacity, whereas higher self-reactivity is associated with enhanced effector function as well as cell cycle entry under physiological conditions. Altogether, our data show that potential effector functions and basal proliferation of CD8 T cells are set by self-reactivity thresholds. PMID:27881740

  7. Efficient generation of functional Schwann cells from adipose-derived stem cells in defined conditions.

    PubMed

    Xie, Songtao; Lu, Fan; Han, Juntao; Tao, Ke; Wang, Hongtao; Simental, Alfred; Hu, Dahai; Yang, Hao

    2017-03-15

    Schwann cells (SCs) are hitherto regarded as the most promising candidates for viable cell-based therapy to peripheral nervous system (PNS) injuries or degenerative diseases. However, the extreme drawbacks of transplanting autologous SCs for clinical applications still represent a significant bottleneck in neural regenerative medicine, mainly owing to the need of sacrificing a functional nerve to generate autologous SCs and the nature of slow expansion of the SCs. Thus, it is of great importance to establish an alternative cell system for the generation of sufficient SCs. Here, we demonstrated that adipose-derived stem cells (ADSCs) of rat robustly give rise to morphological, phenotypic and functional SCs using an optimized protocol. After undergoing a 3-week in vitro differentiation, almost all of treated ADSCs exhibited spindle shaped morphology similar to genuine SCs and expressed SC markers GFAP and S100. Most importantly, apart from acquisition of SC antigenic and biochemical features, the ADSC-derived SCs were functionally identical to native SCs as they possess a potential ability to form myelin, and secret nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and glia-derived neurotrophic factor (GDNF). The current study may provide an ideal strategy for harvesting sufficient SCs for cell-based treatment of various peripheral nerve injuries or disorders.

  8. Conditioned Media from Microvascular Endothelial Cells Cultured in Simulated Microgravity Inhibit Osteoblast Activity

    PubMed Central

    Cazzaniga, Alessandra; Castiglioni, Sara; Maier, Jeanette A. M.

    2014-01-01

    Background and Aims. Gravity contributes to the maintenance of bone integrity. Accordingly, weightlessness conditions during space flight accelerate bone loss and experimental models in real and simulated microgravity show decreased osteoblastic and increased osteoclastic activities. It is well known that the endothelium and bone cells cross-talk and this intercellular communication is vital to regulate bone homeostasis. Because microgravity promotes microvascular endothelial dysfunction, we anticipated that the molecular cross-talk between endothelial cells exposed to simulated microgravity and osteoblasts might be altered. Results. We cultured human microvascular endothelial cells in simulated microgravity using the rotating wall vessel device developed by NASA. Endothelial cells in microgravity show growth inhibition and release higher amounts of matrix metalloproteases type 2 and interleukin-6 than controls. Conditioned media collected from microvascular endothelial cells in simulated microgravity were used to culture human osteoblasts and were shown to retard osteoblast proliferation and inhibit their activity. Discussion. Microvascular endothelial cells in microgravity are growth retarded and release high amounts of matrix metalloproteases type 2 and interleukin-6, which might play a role in retarding the growth of osteoblasts and impairing their osteogenic activity. Conclusions. We demonstrate that since simulated microgravity modulates microvascular endothelial cell function, it indirectly impairs osteoblastic function. PMID:25210716

  9. Influence of growth conditions on the performance of InP nanowire solar cells.

    PubMed

    Cavalli, Alessandro; Cui, Yingchao; Kölling, Sebastian; Verheijen, Marcel A; Plissard, Sebastien R; Wang, Jia; Koenraad, Paul M; Haverkort, Jos E M; Bakkers, Erik P A M

    2016-11-11

    Nanowire based solar cells have attracted great attention due to their potential for high efficiency and low device cost. Photovoltaic devices based on InP nanowires now have characteristics comparable to InP bulk solar cells. A detailed and direct correlation of the influence of growth conditions on performance is necessary to improve efficiency further. We explored the effects of the growth temperature, and of the addition of HCl during growth, on the efficiency of nanowire array based solar cell devices. By increasing HCl, the saturation dark current was reduced, and thereby the nanowire solar cell efficiency was enhanced from less than 1% to 7.6% under AM 1.5 illumination at 1 sun. At the same time, we observed that the solar cell efficiency decreased by increasing the tri-methyl-indium content, strongly suggesting that these effects are carbon related.

  10. Pharyngeal Satellite Cells Undergo Myogenesis Under Basal Conditions and Are Required for Pharyngeal Muscle Maintenance.

    PubMed

    Randolph, Matthew E; Phillips, Brittany L; Choo, Hyo-Jung; Vest, Katherine E; Vera, Yandery; Pavlath, Grace K

    2015-12-01

    The pharyngeal muscles of the nasal, oral, and laryngeal pharynxes are required for swallowing. Pharyngeal muscles are preferentially affected in some muscular dystrophies yet spared in others. Muscle stem cells, called satellite cells, may be critical factors in the development of pharyngeal muscle disorders; however, very little is known about pharyngeal satellite cells (PSC) and their role in pharyngeal muscles. We show that PSC are distinct from the commonly studied hindlimb satellite cells both transcriptionally and biologically. Under basal conditions PSC proliferate, progress through myogenesis, and fuse with pharyngeal myofibers. Furthermore, PSC exhibit biologic differences dependent on anatomic location in the pharynx. Importantly, PSC are required to maintain myofiber size and myonuclear number in pharyngeal myofibers. Together, these results demonstrate that PSC are critical for pharyngeal muscle maintenance and suggest that satellite cell impairment could contribute to pharyngeal muscle pathology associated with various muscular dystrophies and aging.

  11. Influence of growth conditions on the performance of InP nanowire solar cells

    NASA Astrophysics Data System (ADS)

    Cavalli, Alessandro; Cui, Yingchao; Kölling, Sebastian; Verheijen, Marcel A.; Plissard, Sebastien R.; Wang, Jia; Koenraad, Paul M.; Haverkort, Jos E. M.; Bakkers, Erik P. A. M.

    2016-11-01

    Nanowire based solar cells have attracted great attention due to their potential for high efficiency and low device cost. Photovoltaic devices based on InP nanowires now have characteristics comparable to InP bulk solar cells. A detailed and direct correlation of the influence of growth conditions on performance is necessary to improve efficiency further. We explored the effects of the growth temperature, and of the addition of HCl during growth, on the efficiency of nanowire array based solar cell devices. By increasing HCl, the saturation dark current was reduced, and thereby the nanowire solar cell efficiency was enhanced from less than 1% to 7.6% under AM 1.5 illumination at 1 sun. At the same time, we observed that the solar cell efficiency decreased by increasing the tri-methyl-indium content, strongly suggesting that these effects are carbon related.

  12. Influence of conditioning regimens and stem cell sources on donor-type chimerism early after stem cell transplantation.

    PubMed

    Sugita, Junichi; Tanaka, Junji; Hashimoto, Aya; Shiratori, Souichi; Yasumoto, Atsushi; Wakasa, Kentaro; Kikuchi, Misato; Shigematsu, Akio; Miura, Yoko; Tsutsumi, Yutaka; Kondo, Takeshi; Asaka, Masahiro; Imamura, Masahiro

    2008-12-01

    We retrospectively analyzed very early chimerism before and ongoing neutrophil engraftment (days 7, 14, 21, 28) and investigated the influence of conditioning regimens and stem cell sources on donor-type chimerism in 59 Japanese patients who had received allogeneic hematopoietic stem cell transplantation. The percentage of donor-type chimerism increased before engraftment in all patients who achieved engraftment. The average percentage of donor-type chimerism in patients who had received reduced-intensity stem cell transplantation (RIST) with total body irradiation (TBI) was significantly higher than that in patients who had received RIST without TBI (98.8% vs 87.5% on day 21, P<0.01; 99.3% vs 84.3% on day 28, P<0.01). The average percentage of donor-type chimerism after peripheral blood stem cell transplantation was significantly higher than that after bone marrow transplantation on day 7 (81.5% vs 43.1%, P<0.01), and the average percentage of donor-type chimerism after cord blood transplantation was significantly lower on day 14 (55.8% vs 84.8%, P<0.05). Compared with the average percentage of donor-type chimerism in patients who achieved engraftment with each stem cell source, a notable decrease in donor-type chimerism was observed in patients who failed to achieve engraftment. This study suggests that differences in conditioning regimens and stem cell sources should be taken into account when considering donor-type chimerism.

  13. Endothelial cells' biophysical, biochemical, and chromosomal aberrancies in high-glucose condition within the diabetic range.

    PubMed

    Rezabakhsh, Aysa; Nabat, Elahe; Yousefi, Mina; Montazersaheb, Soheila; Cheraghi, Omid; Mehdizadeh, Amir; Fathi, Farzaneh; Movassaghpour, Ali Akbar; Maleki-Dizaji, Nasrin; Rahbarghazi, Reza; Garjani, Alireza

    2017-03-01

    To date, many studies have been conducted to find out the underlying mechanisms of hyperglycemia-induced complications in diabetes mellitus, attributed to the cellular pathologies of different cells-especially endothelial cells. However, there are still many ambiguities and unresolved issues to be clarified. Here, we investigated the alteration in biophysical and biochemical properties in human umbilical vein endothelial cells exposed to a high-glucose concentration (30mM), comparable to glucose content in type 2 diabetes mellitus, over a course of 120 hours. In addition to a reduction in the rate of cell viability and induction of oxidative stress orchestrated by the high-glucose condition, the dynamic of the fatty acid profile-including polyunsaturated, monounsaturated, and saturated fatty acids-was also altered in favor of saturated fatty acids. Genetic imbalances were also detected at chromosomal level in the cells exposed to the abnormal concentration of glucose after 120 hours. Moreover, the number of tip cells (CD31(+) /CD34(+) ) and in vitro tubulogenesis capability negatively diminished in comparison to parallel control groups. We found that diabetic hyperglycemia was associated with a decrease in the cell-cell tight junction and upregulation in vascular endothelial cadherin and zonula occludens (ZO)-1 molecules after 72 and 120 hours of exposure to the abnormal glucose concentration, which resulted in a profound reduction in transendothelial electrical resistance. The surface plasmon resonance analysis of the human umbilical vein endothelial cells immobilized on gold-coated sensor chips confirmed the loosening of the cell to cell intercellular junction as well as stable attachment of each cell to the basal surface. Our findings highlighted the disturbing effects of a diabetic hyperglycemia on either biochemical or biophysical properties of endothelial cells.

  14. Skewing the T-cell repertoire by combined DNA vaccination, host conditioning, and adoptive transfer.

    PubMed

    Jorritsma, Annelies; Bins, Adriaan D; Schumacher, Ton N M; Haanen, John B A G

    2008-04-01

    Approaches for T-cell-based immunotherapy that have shown substantial effects in clinical trials are generally based on the adoptive transfer of high numbers of antigen-specific cells, and the success of these approaches is thought to rely on the high magnitude of the tumor-specific T-cell responses that are induced. In this study, we aimed to develop strategies that also yield a T-cell repertoire that is highly skewed toward tumor recognition but do not rely on ex vivo generation of tumor-specific T cells. To this end, the tumor-specific T-cell repertoire was first expanded by DNA vaccination and then infused into irradiated recipients. Subsequent vaccination of the recipient mice with the same antigen resulted in peak CD8(+) T-cell responses of approximately 50%. These high T-cell responses required the presence of antigen-experienced tumor-specific T cells within the graft because only mice that received cells of previously vaccinated donor mice developed effective responses. Tumor-bearing mice treated with this combined therapy showed a significant delay in tumor outgrowth, compared with mice treated by irradiation or vaccination alone. Furthermore, this antitumor effect was accompanied by an increased accumulation of activated and antigen-specific T cells within the tumor. In summary, the combination of DNA vaccination with host conditioning and adoptive transfer generates a marked, but transient, skewing of the T-cell repertoire toward tumor recognition. This strategy does not require ex vivo expansion of cells to generate effective antitumor immunity and may therefore easily be translated to clinical application.

  15. Dynamics of the mammalian cell cycle in physiological and pathological conditions.

    PubMed

    Gérard, Claude; Goldbeter, Albert

    2016-01-01

    A network of cyclin-dependent kinases (Cdks) controls progression along the successive phases G1, S, G2, and M of the mammalian cell cycle. Deregulations in the expression of molecular components in this network often lead to abusive cell proliferation and cancer. Given the complex nature of the Cdk network, it is fruitful to resort to computational models to grasp its dynamical properties. Investigated by means of bifurcation diagrams, a detailed computational model for the Cdk network shows how the balance between quiescence and proliferation is affected by activators (oncogenes) and inhibitors (tumor suppressors) of cell cycle progression, as well as by growth factors and other external factors such as the extracellular matrix (ECM) and cell contact inhibition. Suprathreshold changes in all these factors can trigger a switch in the dynamical behavior of the network corresponding to a bifurcation between a stable steady state, associated with cell cycle arrest, and sustained oscillations of the various cyclin/Cdk complexes, corresponding to cell proliferation. The model for the Cdk network accounts for the dependence or independence of cell proliferation on serum and/or cell anchorage to the ECM. Such computational approach provides an integrated view of the control of cell proliferation in physiological or pathological conditions. Whether the balance is tilted toward cell cycle arrest or cell proliferation depends on the direction in which the threshold associated with the bifurcation is passed once the cell integrates the multiple signals, internal or external to the Cdk network, that promote or impede progression in the cell cycle.

  16. Effect of substrate storage conditions on the stability of "Smart" films used for mammalian cell applications

    NASA Astrophysics Data System (ADS)

    Bluestein, Blake M.; Reed, Jamie A.; Canavan, Heather E.

    2017-01-01

    When poly(N-isopropyl acrylamide) (pNIPAM) is tethered to a surface, it can induce the spontaneous release of a sheet of mammalian cells. The release of cells is a result of the reversible phase transition the polymer undergoes at its lower critical solution temperature (LCST). Many techniques are used for the deposition of pNIPAM onto cell culture substrates. Previously, we compared two methods of deposition (plasma polymerization, and co-deposition with a sol-gel). We proved that although both were technically appropriate for obtaining thermoresponsive pNIPAM films, the surfaces that were co-deposited with a sol-gel caused some disruption in cell activity. The variation of cell behavior could be due to the delamination of pNIPAM films leaching toxic chemicals into solution. In this work, we assessed the stability of these pNIPAM films by manipulating the storage conditions and analyzing the surface chemistry using X-ray photoelectron spectroscopy (XPS) and contact angle measurements over the amount of time required to obtain confluent cell sheets. From XPS, we demonstrated that ppNIPAM (plasma polymerized NIPAM) films remains stable across all storage conditions while sol-gel deposition show large deviations after 48 h of storage. Cell response of the deposited films was assessed by investigating the cytotoxicity and biocompatibility. The 37 °C and high humidity storage affects sol-gel deposited films, inhibiting normal cell growth and proper thermoresponse of the film. Surface chemistry, thermoresponse and cell growth remained similar for all ppNIPAM surfaces, indicating these substrates are more appropriate for mammalian cell culture applications.

  17. Proliferation and differentiation of bone marrow stromal cells under hypoxic conditions

    SciTech Connect

    Ren Hongying; Cai Huiguo; Han Zhongchao; Yang Renchi; Zhao, Qinjun; Cao Ying; Li Jing; Zhou Cixiang; Liao Lianming; Jia Mingyue; Zhao Qian; Chen Guoqiang . E-mail: chengq@shsmu.edu.cn; Zhao, R.C. |. E-mail: chunhuaz@public.tpt.tj.cn

    2006-08-18

    Low oxygen tension is a potent differentiation inducer of numerous cell types and an effective stimulus of many gene expressions. Here, we described that under 8% O{sub 2}, bone marrow stromal cells (MSCs) exhibited proliferative and morphologic changes. The level of differentiated antigen H-2Dd and the number of G{sub 2}/S/M phase cells increased evidently under 8% O{sub 2} condition. Also, the proportion of wide, flattened, and epithelial-like cells (which were alkaline phosphatase staining positive) in MSCs increased significantly. When cultured in adipogenic medium, there was a 5- to 6-fold increase in the number of lipid droplets under hypoxic conditions compared with that in normoxic culture. We also demonstrated the existence of MSC differentiation under hypoxic conditions by electron microscopy. Expression of Oct4 was inhibited under 8% O{sub 2} condition, but after adipocyte differentiation in normoxic culture and hypoxia-mimicking agents cobalt chloride (CoCl{sub 2}) and deferoxamine mesylate (DFX) treatments, Oct4 was still expressed in MSCs. These results indicate hypoxia accelerates MSC differentiation and hypoxia and hypoxia-mimicking agents exert different effects on MSC differentiation.

  18. Improved biocompatibility of small intestinal submucosa (SIS) following conditioning by human endothelial cells.

    PubMed

    Woods, A M; Rodenberg, E J; Hiles, M C; Pavalko, F M

    2004-02-01

    Small intestinal submucosa (SIS) is a naturally occurring tissue matrix composed of extracellular matrix proteins and various growth factors. SIS is derived from the porcine jejunum and functions as a remodeling scaffold for tissue repair. While SIS has proven to be a useful biomaterial for implants in vivo, problems associated with endothelialization and thrombogenicity of SIS implants may limit its vascular utility. The goal of this study was to determine if the biological properties of SIS could be improved by growing human umbilical vein endothelial cells (HUVEC) on SIS and allowing these cells to deposit human basement membrane proteins on the porcine substrate to create what we have called "conditioned" SIS (c-SIS). Using an approach in which HUVEC were grown for 2 weeks on SIS and then removed via a technique that leaves behind an intact basement membrane, we hypothesized that the surface properties of SIS might be improved. We found that when re-seeded on c-SIS, HUVEC exhibited enhanced organization of cell junctions and had increased metabolic activity compared to cells on native SIS (n-SIS). Furthermore, HUVEC grown on c-SIS released lower amounts of the pro-inflammatory prostaglandin PGI2 into the media compared to cells grown on n-SIS. Additionally, we found that adhesion of resting or activated human platelets to c-SIS was significantly decreased compared to n-SIS suggesting that, in addition to improved cell growth characteristics, conditioning SIS with human basement membrane proteins might decrease its thrombogenic potential. In summary, conditioning of porcine SIS by human endothelial cells improves key biological properties of the material that may improve its usefulness as remodeling scaffold for tissue repair. Identification of critical modifications of SIS by human endothelial cells should help guide future efforts to develop more biocompatible vascular grafts.

  19. Critical elements in the development of cell therapy potency assays for ischemic conditions.

    PubMed

    Porat, Yael; Abraham, Eytan; Karnieli, Ohad; Nahum, Sagi; Woda, Juliana; Zylberberg, Claudia

    2015-07-01

    A successful potency assay for a cell therapy product (CTP) used in the treatment of ischemic conditions should quantitatively measure relevant biological properties that predict therapeutic activity. This is especially challenging because of numerous degrees of complexity stemming from factors that include a multifactorial complex mechanism of action, cell source, inherent cell characteristics, culture method, administration mode and the in vivo conditions to which the cells are exposed. The expected biological function of a CTP encompasses complex interactions that range from a biochemical, metabolic or immunological activity to structural replacement of damaged tissue or organ. Therefore, the requirements for full characterization of the active substance with respect to biological function could be taxing. Moreover, the specific mechanism of action is often difficult to pinpoint to a specific molecular entity; rather, it is more dependent on the functionality of the cellular components acting in a in a multifactorial fashion. In the case of ischemic conditions, the cell therapy mechanism of action can vary from angiogenesis, vasculogenesis and arteriogenesis that may activate different pathways and clinical outcomes. The CTP cellular attributes with relation to the suggested mechanism of action can be used for the development of quantitative and reproducible analytical potency assays. CTPs selected and released on the basis of such potency assays should have the highest probability of providing meaningful clinical benefit for patients. This White Paper will discuss and give examples for key elements in the development of a potency assay for treatment of ischemic disorders treated by the use of CTPs.

  20. Derivation of transgene-free human induced pluripotent stem cells from human peripheral T cells in defined culture conditions.

    PubMed

    Kishino, Yoshikazu; Seki, Tomohisa; Fujita, Jun; Yuasa, Shinsuke; Tohyama, Shugo; Kunitomi, Akira; Tabei, Ryota; Nakajima, Kazuaki; Okada, Marina; Hirano, Akinori; Kanazawa, Hideaki; Fukuda, Keiichi

    2014-01-01

    Recently, induced pluripotent stem cells (iPSCs) were established as promising cell sources for revolutionary regenerative therapies. The initial culture system used for iPSC generation needed fetal calf serum in the culture medium and mouse embryonic fibroblast as a feeder layer, both of which could possibly transfer unknown exogenous antigens and pathogens into the iPSC population. Therefore, the development of culture systems designed to minimize such potential risks has become increasingly vital for future applications of iPSCs for clinical use. On another front, although donor cell types for generating iPSCs are wide-ranging, T cells have attracted attention as unique cell sources for iPSCs generation because T cell-derived iPSCs (TiPSCs) have a unique monoclonal T cell receptor genomic rearrangement that enables their differentiation into antigen-specific T cells, which can be applied to novel immunotherapies. In the present study, we generated transgene-free human TiPSCs using a combination of activated human T cells and Sendai virus under defined culture conditions. These TiPSCs expressed pluripotent markers by quantitative PCR and immunostaining, had a normal karyotype, and were capable of differentiating into cells from all three germ layers. This method of TiPSCs generation is more suitable for the therapeutic application of iPSC technology because it lowers the risks associated with the presence of undefined, animal-derived feeder cells and serum. Therefore this work will lead to establishment of safer iPSCs and extended clinical application.

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

    PubMed Central

    Vesole, D H; Moore, G E

    1980-01-01

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

  2. Increased performance of hydrogen production in microbial electrolysis cells under alkaline conditions.

    PubMed

    Rago, Laura; Baeza, Juan A; Guisasola, Albert

    2016-06-01

    This work reports the first successful enrichment and operation of alkaline bioelectrochemical systems (microbial fuel cells, MFC, and microbial electrolysis cells, MEC). Alkaline (pH=9.3) bioelectrochemical hydrogen production presented better performance (+117%) compared to conventional neutral conditions (2.6 vs 1.2 litres of hydrogen gas per litre of reactor per day, LH2·L(-1)REACTOR·d(-1)). Pyrosequencing results of the anodic biofilm showed that while Geobacter was mainly detected under conventional neutral conditions, Geoalkalibacter sp. was highly detected in the alkaline MFC (21%) and MEC (48%). This is the first report of a high enrichment of Geoalkalibacter from an anaerobic mixed culture using alkaline conditions in an MEC. Moreover, Alkalibacter sp. was highly present in the anodic biofilm of the alkaline MFC (37%), which would indicate its potentiality as a new exoelectrogen.

  3. Developmental significance of cyanide-resistant respiration under stressed conditions: experiments in Dictyostelium cells.

    PubMed

    Kimura, Kei; Kuwayama, Hidekazu; Amagai, Aiko; Maeda, Yasuo

    2010-09-01

    We have previously reported that benzohydroxamic acid (BHAM), a potent inhibitor of cyanide (CN)-resistant respiration mediated by alternative oxidase (AOX), induces formation of unique cell masses (i.e., stalk-like cells with a large vacuole and thick cell wall) in starved Dictyostelium cells. Unexpectedly, however, aox-null cells prepared by homologous recombination exhibited normal development under normal culture conditions on agar, indicating that BHAM-induced stalk formation is not solely attributable to inhibition of CN-resistant respiration. This also suggests that a series of pharmacological approaches in the field of life science has serious limitations. Under stress (e.g., in submerged culture), starved aox-null cells exhibited slightly delayed aggregation compared with parental Ax-2 cells; most cells remained as loose aggregates even after prolonged incubation. Also, the developmental defects of aox-null cells became more marked upon incubation for 30 min just after starvation in the presence of ≥ 1.75 mmol/L H(2)O(2). This seems to indicate that CN-resistant respiration could mitigate cellular damage through reactive oxygen species (ROS), because AOX has a potential role in reduction of ROS production. Starved aox-null cells did not develop in the presence of 5 mmol/L KCN (which completely inhibited the conventional cytochrome-mediated respiration) and remained as non-aggregated single cells on agar even after prolonged incubation. Somewhat surprisingly, however, parental Ax-2 cells were found to develop normally, forming fruiting bodies even in the presence of 10 mmol/L KCN. Taken together, these results suggest that CN-resistant respiration might compensate for the production of adenosine tri-phosphate via oxidative phosphorylation.

  4. Neurosphere and adherent culture conditions are equivalent for malignant glioma stem cell lines

    PubMed Central

    Reyner, Karina; Deleyrolle, Loic; Millette, Sebastien; Azari, Hassan; Day, Bryan W.; Stringer, Brett W.; Boyd, Andrew W.; Johns, Terrance G.; Blot, Vincent; Duggal, Rohit; Reynolds, Brent A.

    2015-01-01

    Certain limitations of the neurosphere assay (NSA) have resulted in a search for alternative culture techniques for brain tumor-initiating cells (TICs). Recently, reports have described growing glioblastoma (GBM) TICs as a monolayer using laminin. We performed a side-by-side analysis of the NSA and laminin (adherent) culture conditions to compare the growth and expansion of GBM TICs. GBM cells were grown using the NSA and adherent culture conditions. Comparisons were made using growth in culture, apoptosis assays, protein expression, limiting dilution clonal frequency assay, genetic affymetrix analysis, and tumorigenicity in vivo. In vitro expansion curves for the NSA and adherent culture conditions were virtually identical (P=0.24) and the clonogenic frequencies (5.2% for NSA vs. 5.0% for laminin, P=0.9) were similar as well. Likewise, markers of differentiation (glial fibrillary acidic protein and beta tubulin III) and proliferation (Ki67 and MCM2) revealed no statistical difference between the sphere and attachment methods. Several different methods were used to determine the numbers of dead or dying cells (trypan blue, DiIC, caspase-3, and annexin V) with none of the assays noting a meaningful variance between the two methods. In addition, genetic expression analysis with microarrays revealed no significant differences between the two groups. Finally, glioma cells derived from both methods of expansion formed large invasive tumors exhibiting GBM features when implanted in immune-compromised animals. A detailed functional, protein and genetic characterization of human GBM cells cultured in serum-free defined conditions demonstrated no statistically meaningful differences when grown using sphere (NSA) or adherent conditions. Hence, both methods are functionally equivalent and remain suitable options for expanding primary high-grade gliomas in tissue culture. PMID:25806119

  5. Proinflammatory interleukins' production by adipose tissue-derived mesenchymal stromal cells: the impact of cell culture conditions and cell-to-cell interaction.

    PubMed

    Andreeva, Elena; Andrianova, Irina; Rylova, Julia; Gornostaeva, Aleksandra; Bobyleva, Polina; Buravkova, Ludmila

    2015-08-01

    The impact of culture conditions and interaction with activated peripheral blood mononuclear cells on the interleukin (IL) gene expression profile and proinflammatory IL-6 and IL-8 production by adipose-derived stromal cells (ASCs) was investigated. A microarray analysis revealed a wide range of IL genes either under standard (20%) or hypoxic (5%) O2 concentrations, some highly up-regulated at hypoxia. IL-6 and IL-8 production was inversely dependent on cell culture density. In early (first-third) passages, IL-6 and IL-8 concentration was higher at 20% O2 and in late (8th-12th) passages under 5% O2. Interaction between ASCs and mononuclear cells in indirect setting was accompanied with a significant decrease of IL-6 and did not result in the elevation of IL-8 concentration. Thereby, the production of proinflammatory interleukins (IL-6 and IL-8) may be affected by the ASC intrinsic features (density in culture, and duration of expansion), as well as by microenvironmental factors, such as hypoxia and the presence of blood-borne cells. These data are important for elucidating ASC paracrine activity regulation in vitro. They would also be on demand for optimisation of the cell therapy protocols, based on the application of ASC biologically active substances. SIGNIFICANCE PARAGRAPH: Ex vivo expansion is widely used for increasing the number of adipose-derived stromal cells (ASCs) and improving of their quality. The present study was designed to elucidate the particular factors influencing the interleukin production in ASCs. The presented data specified the parameters (i.e. cell density, duration of cultivation, hypoxia, etc.) that should be taken in mind when ASCs are intended to be used in protocols implying their paracrine activity. These data would be of considerable interest for researchers and clinicians working in the biomedical science.

  6. The effect of neurosphere culture conditions on the cellular metabolism of glioma cells.

    PubMed

    Kahlert, Ulf Dietrich; Koch, Katharina; Suwala, Abigail Kora; Hartmann, Rudolf; Cheng, Menglin; Maciaczyk, Donata; Willbold, Dieter; Eberhart, Charles G; Glunde, Kristine; Maciaczyk, Jarek

    2015-01-01

    Malignant gliomas, with an average survival time of 16-19 months after initial diagnosis, account for one of the most lethal tumours overall. Current standards in patient care provide only unsatisfying strategies in diagnostic and treatment for high-grade gliomas. Here we describe metabolic phenomena in the choline and glycine network associated with stem cell culture conditions in the classical glioma cell line U87. Using high-resolution proton magnetic resonance spectroscopy of cell culture metabolic extracts we compare the metabolic composition of U87 chronically propagated as adherent culture in medium supplemented with serum to serum-free neurosphere growth. We found that the switch to neurosphere growth, besides the increase of cells expressing the putative glioma stem cell marker CD133, modulated a number of intracellular metabolites including choline, creatine, glycine, and myo-inositol that have been previously reported as potential diagnostic markers in various tumours. These findings highlight the critical influence of culture conditions on glioma cell metabolism, and therefore particular caution should be drawn to the use of in vitro system research in order to investigate cancer metabolism.

  7. Transporting Cells in Semi-Solid Gel Condition and at Ambient Temperature

    PubMed Central

    Wang, Junjian; Chen, Peng; Xu, Jianzhen; Zou, June.X; Wang, Haibin; Chen, Hong-Wu

    2015-01-01

    Mammalian cells including human cancer cells are usually transported in cryovials on dry ice or in a liquid nitrogen vapor shipping vessel between different places at long distance. The hazardous nature of dry ice and liquid nitrogen, and the associated high shipping cost strongly limit their routine use. In this study, we tested the viability and properties of cells after being preserved or shipped over long distance in Matrigel mixture for different days. Our results showed that cells mixed with Matrigel at suitable ratios maintained excellent viability (>90%) for one week at room temperature and preserved the properties such as morphology, drug sensitivity and metabolism well, which was comparable to cells cryopreserved in liquid nitrogen. We also sent cells in the Matrigel mixture via FedEx service to different places at ambient temperature. Upon arrival, it was found that over 90% of the cells were viable and grew well after replating. These data collectively suggested that our Matrigel-based method was highly convenient for shipping live cells for long distances in semi-solid gel condition and at ambient temperature. PMID:26098554

  8. Optical examination of cell culture in bioreactors creating simulated in vivo conditions

    NASA Astrophysics Data System (ADS)

    Rolfe, Peter J.

    2005-01-01

    Cell culture using bioreactors is a vital part of Cellular and Tissue Engineering. Bioreactor design continues to advance, in order to allow control over physical and chemical parameters as well as continuous assessment of cell behaviour, gene expression, and tissue formation and growth. Measurement or monitoring of many such parameters or features can be achieved with optical techniques. The current aim of cell culture is to re-create in vivo conditions and in order to achieve this control of the chemical environment is required and some cell types must be subjected to shear stress and/or axial loads. For creating tissue engineered cartilage chondrocytes are cultured within a biodegradable scaffold. Influences of cyclic loading and of oxygen supply on phenotype are studied. Vascular endothelial cells are subjected to fluid shear stress and the influence on prostacyclin production is measured. Optical interrogation of culture fluid, attached cells, cells in suspension and tissue constructs is carried out using a combination of spectrophotometry, analysis of scattering, and chemical sensing. Insertion of sensing probes within the culture vessel presents problems of protein adsorption to sensing surfaces. Approaches based on cell membrane mimicry are being evaluated for their potential to overcome this problem. Sensors based on immobilised fluorophores and chromaphores within either wall-mounted membranes or within optical fibres are assessed. Culture fluid turbidity is evaluated with scattering determinations and circulating glucose concentration is measured spectrophotometrically. Formed tissue is interrogated with NIR radiation and in the future will include the use of OCT.

  9. Conditional Cripto overexpression in satellite cells promotes myogenic commitment and enhances early regeneration

    PubMed Central

    Prezioso, Carolina; Iaconis, Salvatore; Andolfi, Gennaro; Zentilin, Lorena; Iavarone, Francescopaolo; Guardiola, Ombretta; Minchiotti, Gabriella

    2015-01-01

    Skeletal muscle regeneration mainly depends on satellite cells, a population of resident muscle stem cells. Despite extensive studies, knowledge of the molecular mechanisms underlying the early events associated with satellite cell activation and myogenic commitment in muscle regeneration remains still incomplete. Cripto is a novel regulator of postnatal skeletal muscle regeneration and a promising target for future therapy. Indeed, Cripto is expressed both in myogenic and inflammatory cells in skeletal muscle after acute injury and it is required in the satellite cell compartment to achieve effective muscle regeneration. A critical requirement to further explore the in vivo cellular contribution of Cripto in regulating skeletal muscle regeneration is the possibility to overexpress Cripto in its endogenous configuration and in a cell and time-specific manner. Here we report the generation and the functional characterization of a novel mouse model for conditional expression of Cripto, i.e., the Tg:DsRedloxP/loxPCripto-eGFP mice. Moreover, by using a satellite cell specific Cre-driver line we investigated the biological effect of Cripto overexpression in vivo, and provided evidence that overexpression of Cripto in the adult satellite cell compartment promotes myogenic commitment and differentiation, and enhances early regeneration in a mouse model of acute injury. PMID:26052513

  10. Conditional Cripto overexpression in satellite cells promotes myogenic commitment and enhances early regeneration.

    PubMed

    Prezioso, Carolina; Iaconis, Salvatore; Andolfi, Gennaro; Zentilin, Lorena; Iavarone, Francescopaolo; Guardiola, Ombretta; Minchiotti, Gabriella

    2015-01-01

    Skeletal muscle regeneration mainly depends on satellite cells, a population of resident muscle stem cells. Despite extensive studies, knowledge of the molecular mechanisms underlying the early events associated with satellite cell activation and myogenic commitment in muscle regeneration remains still incomplete. Cripto is a novel regulator of postnatal skeletal muscle regeneration and a promising target for future therapy. Indeed, Cripto is expressed both in myogenic and inflammatory cells in skeletal muscle after acute injury and it is required in the satellite cell compartment to achieve effective muscle regeneration. A critical requirement to further explore the in vivo cellular contribution of Cripto in regulating skeletal muscle regeneration is the possibility to overexpress Cripto in its endogenous configuration and in a cell and time-specific manner. Here we report the generation and the functional characterization of a novel mouse model for conditional expression of Cripto, i.e., the Tg:DsRed (loxP/loxP) Cripto-eGFP mice. Moreover, by using a satellite cell specific Cre-driver line we investigated the biological effect of Cripto overexpression in vivo, and provided evidence that overexpression of Cripto in the adult satellite cell compartment promotes myogenic commitment and differentiation, and enhances early regeneration in a mouse model of acute injury.

  11. Regenerative and reparative effects of human chorion-derived stem cell conditioned medium on photo-aged epidermal cells.

    PubMed

    Li, Qiankun; Chen, Yan; Ma, Kui; Zhao, Along; Zhang, Cuiping; Fu, Xiaobing

    2016-01-01

    Epidermal cells are an important regenerative source for skin wound healing. Aged epidermal cells have a low ability to renew themselves and repair skin injury. Ultraviolet (UV) radiation, particularly UVB, can cause photo-aging of the skin by suppressing the viability of human epidermal cells. A chorion-derived stem cell conditioned medium (CDSC-CNM) is thought to have regenerative properties. This study aimed to determine the regenerative effects of CDSC-CNM on UVB-induced photo-aged epidermal cells. Epidermal cells were passaged four times and irradiated with quantitative UVB, and non-irradiated cells served as a control group. Cells were then treated with different concentrations of CDSC-CNM. Compared to the non-irradiated group, the proliferation rates and migration rates of UVB-induced photo-aged epidermal cells significantly decreased (p < 0.05) with increasing intracellular radical oxygen species (ROS) generation and DNA damage. After treatment with CDSC-CNM, photo-aged epidermal cells significantly improved their viability, and their ROS generation and DNA damage decreased. The secretory factors in CDSC-CNM, including epidermal growth factor (EGF), transforming growth factor-β (TGF-β), interleukin (IL)-6, and IL-8 and the related signaling pathway protein levels, increased compared to the control medium (CM). The potential regenerative and reparative effects of CDSC-CNM indicate that it may be a candidate material for the treatment of prematurely aged skin. The functions of the secretory factors and the mechanisms of CDSC-CNM therapy deserve further attention.

  12. T Cells Enhance Stem-Like Properties and Conditional Malignancy in Gliomas

    PubMed Central

    Irvin, Dwain K.; Jouanneau, Emmanuel; Duvall, Gretchen; Zhang, Xiao-xue; Zhai, Yuying; Sarayba, Danielle; Seksenyan, Akop; Panwar, Akanksha; Black, Keith L.; Wheeler, Christopher J.

    2010-01-01

    Background Small populations of highly tumorigenic stem-like cells (cancer stem cells; CSCs) can exist within, and uniquely regenerate cancers including malignant brain tumors (gliomas). Many aspects of glioma CSCs (GSCs), however, have been characterized in non-physiological settings. Methods We found gene expression similarity superiorly defined glioma “stemness”, and revealed that GSC similarity increased with lower tumor grade. Using this method, we examined stemness in human grade IV gliomas (GBM) before and after dendritic cell (DC) vaccine therapy. This was followed by gene expression, phenotypic and functional analysis of murine GL26 tumors recovered from nude, wild-type, or DC-vaccinated host brains. Results GSC similarity was specifically increased in post-vaccine GBMs, and correlated best to vaccine-altered gene expression and endogenous anti-tumor T cell activity. GL26 analysis confirmed immune alterations, specific acquisition of stem cell markers, specifically enhanced sensitivity to anti-stem drug (cyclopamine), and enhanced tumorigenicity in wild-type hosts, in tumors in proportion to anti-tumor T cell activity. Nevertheless, vaccine-exposed GL26 cells were no more tumorigenic than parental GL26 in T cell-deficient hosts, though they otherwise appeared similar to GSCs enriched by chemotherapy. Finally, vaccine-exposed GBM and GL26 exhibited relatively homogeneous expression of genes expressed in progenitor cells and/or differentiation. Conclusions T cell activity represents an inducible physiological process capable of proportionally enriching GSCs in human and mouse gliomas. Stem-like gliomas enriched by strong T cell activity, however, may differ from other GSCs in that their stem-like properties may be disassociated from increased tumor malignancy and heterogeneity under specific host immune conditions. PMID:20539758

  13. Protein secretory patterns of rat Sertoli and peritubular cells are influenced by culture conditions

    SciTech Connect

    Kierszenbaum, A.L.; Crowell, J.A.; Shabanowitz, R.B.; DePhilip, R.M.; Tres, L.L.

    1986-08-01

    An approach combining two-dimensional gel electrophoresis and autoradiography was used to correlate patterns of secretory proteins in cultures of Sertoli and peritubular cells with those observed in the incubation medium from segments of seminiferous tubules. Sertoli cells in culture and in seminiferous tubules secreted three proteins designated S70 (Mr 72,000-70,000), S45 (Mr 45,000), and S35 (Mr 35,000). Cultured Sertoli and peritubular cells and incubated seminiferous tubules secreted two proteins designated SP1 (Mr 42,000) and SP2 (Mr 50,000). SP1 and S45 have similar Mr but differ from each other in isoelectric point (pI). Cultured peritubular cells secreted a protein designated P40 (Mr 40,000) that was also seen in intact seminiferous tubules but not in seminiferous tubules lacking the peritubular cell wall. However, a large number of high-Mr proteins were observed only in the medium of cultured peritubular cells but not in the incubation medium of intact seminiferous tubules. Culture conditions influence the morphology and patterns of protein secretion of cultured peritubular cells. Peritubular cells that display a flat-stellate shape transition when placed in culture medium free of serum (with or without hormones and growth factors), accumulate various proteins in the medium that are less apparent when these cells are maintained in medium supplemented with serum. Two secretory proteins stimulated by follicle-stimulating hormone (FSH) (designated SCm1 and SCm2) previously found in the medium of cultured Sertoli cells, were also observed in the incubation medium of seminiferous tubular segments stimulated by FSH. Results of this study show that, although cultured Sertoli and peritubular cells synthesize and secrete proteins also observed in segments of incubated seminiferous tubules anther group of proteins lacks seminiferous tubular correlates.

  14. PPAR-delta promotes survival of breast cancer cells in harsh metabolic conditions

    PubMed Central

    Wang, X; Wang, G; Shi, Y; Sun, L; Gorczynski, R; Li, Y-J; Xu, Z; Spaner, D E

    2016-01-01

    Expression of the nuclear receptor peroxisome proliferator activated receptor delta (PPARδ) in breast cancer cells is negatively associated with patient survival, but the underlying mechanisms are not clear. High PPARδ protein levels in rat breast adenocarcinomas were found to be associated with increased growth in soft agar and mice. Transgenic expression of PPARδ increased the ability of human breast cancer cell lines to migrate in vitro and form lung metastases in mice. PPARδ also conferred the ability to grow in exhausted tissue culture media and survive in low-glucose and other endoplasmic reticulum stress conditions such as hypoxia. Upregulation of PPARδ by glucocorticoids or synthetic agonists also protected human breast cancer cells from low glucose. Survival in low glucose was related to increased antioxidant defenses mediated in part by catalase and also to late AKT phosphorylation, which is associated with the prolonged glucose-deprivation response. Synthetic antagonists reversed the survival benefits conferred by PPARδ in vitro. These findings suggest that PPARδ conditions breast cancer cells to survive in harsh microenvironmental conditions by reducing oxidative stress and enhancing survival signaling responses. Drugs that target PPARδ may have a role in the treatment of breast cancer. PMID:27270614

  15. Osmolytes responsible for volume reduction under isosmotic or hypoosmotic conditions in Barnacle muscle cells.

    PubMed

    Peña-Rasgado, C; Pierce, S K; Rasgado-Flores, H

    2001-07-01

    In numerous animal cells, experimental manipulations that increase the intracellular free Ca2+ concentration induce cell volume reduction. This may occur under isosmotic conditions, e.g. when external Ca2+ (Ca(o)) is replaced by Mg2+ (42) or during exposure to hypoosmotic conditions (i.e. regulatory volume decrease, RVD) in the presence of Ca(o). We determined the osmolytes responsible for volume reduction under isosmotic and hypoosmotic conditions in barnacle muscle cells. Organic osmolytes (i.e. free amino acids and methylamines) and inorganic ions accounted for approximately 78% and 22% of the intracellular isosmotic activity, respectively. Isosmotic Ca(o) removal induced a net loss of KCI (with a ratio of 1K:1Cl) and free amino acids (FAA, mainly glycine and taurine). During RVD. the same ions (but in a proportion of 2K:1Cl) and FAA were lost. Since RVD was accompanied by extracellular alkalinization, the 2K:1Cl loss may be explained by the presence of a K+/H+ exchanger (or K+-OH- co-transporter) or Cl-/OH- exchanger. The lack of RVD in the absence of Ca(o) cannot be attributed to the loss of intracellular osmolytes during isosmotic Ca(o) removal because addition of Ca(o) during cell swelling promoted RVD.

  16. Plasticity in variation of xylem and phloem cell characteristics of Norway spruce under different local conditions

    PubMed Central

    Gričar, Jožica; Prislan, Peter; de Luis, Martin; Gryc, Vladimír; Hacurová, Jana; Vavrčík, Hanuš; Čufar, Katarina

    2015-01-01

    There is limited information on intra-annual plasticity of secondary tissues of tree species growing under different environmental conditions. To increase the knowledge about the plasticity of secondary growth, which allows trees to adapt to specific local climatic regimes, we examined climate–radial growth relationships of Norway spruce [Picea abies (L.) H. Karst.] from three contrasting locations in the temperate climatic zone by analyzing tree-ring widths for the period 1932–2010, and cell characteristics in xylem and phloem increments formed in the years 2009–2011. Variation in the structure of xylem and phloem increments clearly shows that plasticity in seasonal dynamics of cambial cell production and cell differentiation exists on xylem and phloem sides. Anatomical characteristics of xylem and phloem cells are predominantly site-specific characteristics, because they varied among sites but were fairly uniform among years in trees from the same site. Xylem and phloem tissues formed in the first part of the growing season seemed to be more stable in structure, indicating their priority over latewood and late phloem for tree performance. Long-term climate and radial growth analyses revealed that growth was in general less dependent on precipitation than on temperature; however, growth sensitivity to local conditions differed among the sites. Only partial dependence of radial growth of spruce on climatic factors on the selected sites confirms its strategy to adapt the structure of wood and phloem increments to function optimally in local conditions. PMID:26442044

  17. A semi-empirical glycosylation model of a camelid monoclonal antibody under hypothermia cell culture conditions.

    PubMed

    Aghamohseni, Hengameh; Spearman, Maureen; Ohadi, Kaveh; Braasch, Katrin; Moo-Young, Murray; Butler, Michael; Budman, Hector M

    2017-03-11

    The impact of cell culture environment on the glycan distribution of a monoclonal antibody (mAb) has been investigated through a combination of experiments and modeling. A newly developed CHO DUXB cell line was cultivated at two levels of initial Glutamine (Gln) concentrations (0, 4 mM) and incubation temperatures of (33 and 37 °C) in batch operation mode. Hypothermia was applied either through the entire culture duration or only during the post-exponential phase. Beyond reducing cell growth and increasing productivity, hypothermia significantly altered the galactosylation index profiles as compared to control conditions. A novel semi-empirical dynamic model was proposed for elucidating the connections between the extracellular cell culture conditions to galactosylation index. The developed model is based on a simplified balance of nucleotides sugars and on the correlation between sugars' levels to the galactosylation index (GI). The model predictions were found to be in a good agreement with the experimental data. The proposed empirical model is expected to be useful for controlling the glycoprofiles by manipulating culture conditions.

  18. Changes in cell wall architecture of wheat coleoptiles grown under continuous hypergravity conditions

    NASA Astrophysics Data System (ADS)

    Wakabayashi, K.; Soga, K.; Kamisaka, S.; Hoson, T.

    Modifications of cell wall structure of wheat coleoptiles in response to continuous hypergravity (300 g) treatment were investigated. Length of coleoptiles exposed to hypergravity for 2-4 days from germination stage was 60-70% of that of 1 g control. The net amounts of cell wall polysaccharides, such as hemicellulose and cellulose, of hypergravity-treated coleoptiles increased as much as those of 1 g control coleoptiles during the incubation period. As a result, the levels of cell wall polysaccharides per unit length of coleoptile, which mean the thickness of cell walls, largely increased under hypergravity conditions. Particularly, the amounts of hemicellulosic polymers with middle molecular mass (0.2-1 MDa) largely increased from day 2 to 3 under hypergravity conditions. The major sugar components of the hemicellulose fraction are arabinose, xylose and glucose. The ratios of arabinose and xylose to glucose were higher in hypergravity-treated coleoptiles than in control coleoptiles. The fractionation of hemicellulosic polymers into the neutral and acidic polymers by the anion-exchange column showed that the levels of acidic polymers (mainly composed of arabinoxylans) in cell walls of hypergravity-treated coleoptiles were higher than those of control coleoptiles. In addition to wall polysaccharides, the amounts of cell wall-bound phenolics, such as ferulic acid and diferulic acid, substantially increased during the incubation period both in 1 g control and hypergravity-treated coleoptiles. Especially, the levels of diferulic acid which cross-links hemicellulosic polymers were higher in hypergravity-treated coleoptiles than in control coleoptiles during the incubation period. These results suggest that hypergravity stimuli from the germination stage bias the type of synthesized hemicellulosic polysaccharides, although they do not restrict the net synthesis of cell wall constituents in wheat coleoptiles. The stimulation of the synthesis of arabinoxylans and of the

  19. Inhibitory effects of autologous γ-irradiated cell conditioned medium on osteoblasts in vitro.

    PubMed

    Li, Xu-Fang; Zhu, Guo-Ying; Wang, Jian-Ping; Wang, Yu

    2015-07-01

    Skeletal complications from radiation therapy have been reported in patients with breast, brain and pelvic cancer, and types of blood cancer. However, it remains to be elucidated whether localized radiotherapy may result in systemic adverse effects on the unirradiated skeleton through an abscopal mechanism. The present study investigated the abscopal effect of radiation on osteoblasts mediated by autologous γ-irradiated cell conditioned medium. Osteoblasts obtained from calvarial bones were incubated with irradiated cell conditioned medium (ICCM) and changes in cell viability, alkaline phosphatase (ALP) activity, mineralization ability, cell apoptosis and the gene expression levels of ALP, osteocalcin (BGP), osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand (RANKL) and caspase 3 were observed. Notably, ICCM regulated osteoblast function, inhibiting viability and differentiation, resulting in apoptosis or cell death. ICCM at 10 or 20%, from osteoblasts irradiated with 10 Gy γ-rays, significantly inhibited the proliferation of osteoblastic cells (P<0.001). In addition, an increase in apoptosis was noted in the osteoblasts incubated with ICCM at 40% with increasing doses of radiation, accompanied by an upregulation in the mRNA expression of caspase 3. In addition, ICCM at 20% inhibited the ALP activity in the 5 and 10 Gy groups and osteoblast mineralization, particularly at 10 Gy ICCM. Additionally, the mRNA expression levels of ALP, BGP, OPG and RANKL of the cells treated with ICCM at 20% were downregulated significantly compared with those treated with medium from unirradiated cells. The present study provided novel evidence to elucidate radiation-therapy-associated side effects on the skeleton.

  20. The normal flora may contribute to the quantitative preponderance of myeloid cells under physiological conditions.

    PubMed

    Liang, Shi; LiHua, Hu

    2011-01-01

    Under physiological conditions, the innate immune cells derived from myeloid lineage absolutely outnumber the lymphoid cells. At present, two theories are attributed to the maintenance of haemopoiesis: the asymmetric cell division and the bone marrow hematopoietic microenvironment or "niche". However, the former only explains the self-renewal of haemopoietic stem cell (HSC) and the start of haemopoietic differentiation but fails to address the inducers of cell fate decisions; the latter has to admit that the hematopoietic cytokines, despite their significance in the maintenance of haemopoiesis, have no specific effect on lineage commitment. Given these flaws, the advantageous mechanism of myeloid haemopoiesis has not yet been uncovered in the current theories. The discoveries that bacterial components (lipopolysaccharide, LPS) and intestinal decontamination affect the mobilization of HSC trigger the interest in normal flora, which together with their components may have an effect on haemopoiesis. In the experiments in dogs and mice, researchers documented that the generation of myeloid cells has undergone changes in the bone marrow and periphery when antibiotics are used to regulate the normal intestinal flora and the concentration of its components. However, the same changes are not involved in lymphoid cells. Therefore, we hypothesize that in human body normal flora and its components are a driving force to maintain myeloid haemopoiesis under physiological conditions. To account for the selectiveness in haemopoiesis, these facts should be taken into consideration, such as HSC and mesenchymal stem cells (MSC) functionally expressed pattern recognition receptors (PRR), and both of them can self-migrate or be recruited by normal flora or its components into periphery. Dynamically monitoring the myeloid haemopoiesis may provide an important complementary program that precludes the abuse of antibiotics, which prevents diseases triggered by the imbalance of normal

  1. Diabetic conditions promote binding of monocytes to vascular smooth muscle cells and their subsequent differentiation.

    PubMed

    Meng, Li; Park, Jehyun; Cai, Qiangjun; Lanting, Linda; Reddy, Marpadga A; Natarajan, Rama

    2010-03-01

    Diabetes is associated with significantly accelerated rates of atherosclerosis, key features of which include the presence of excessive macrophage-derived foam cells in the subendothelial space. We examined the hypothesis that enhanced monocyte-vascular smooth muscle cell (VSMC) interactions leading to subendothelial monocyte retention and differentiation to macrophages under diabetic conditions may be underlying mechanisms. Human aortic VSMCs (HVSMCs) treated with diabetic stimuli high glucose (HG) or S100B, a ligand of the receptor for advanced glycation end products, exhibited significantly increased binding of THP-1 monocytic cells. Diabetic stimuli increased the expression of the adhesive chemokine fractalkine (FKN) in HVSMCs. Pretreatment of HVSMCs with FKN or monocyte chemoattractant protein-1 (MCP-1) neutralizing antibodies significantly inhibited monocyte-VSMC binding, whereas monocytes treated with FKN showed enhanced binding to VSMC. Mouse aortic VSMCs (MVSMCs) derived from type 2 diabetic db/db mice exhibited significantly increased FKN levels and binding to mouse WEHI78/24 monocytic cells relative to nondiabetic control db/+ cells. The enhanced monocyte binding in db/db cells was abolished by both FKN and MCP-1 antibodies. Endothelium-denuded aortas from db/db mice and streptozotocin-induced diabetic mice also exhibited enhanced FKN expression and monocyte binding, relative to respective controls. Coculture with HVSMCs increased CD36 expression in THP-1 cells, and this was significantly augmented by treatment of HVSMCs with S100B or HG. CD36 mRNA and protein levels were also significantly increased in WEHI78/24 cells after coincubation with db/db MVSMCs relative to control MVSMCs. These results demonstrate that diabetic conditions may accelerate atherosclerosis by inducing key chemokines in the vasculature that promote VSMC-monocyte interactions, subendothelial monocyte retention, and differentiation.

  2. Osmotic properties of internally perfused barnacle muscle cells. I. Isosmotic conditions.

    PubMed

    Bitner, J B; Peña-Rasgado, C; Ruiz, J; Cardona, J; Rasgado-Flores, H

    2001-07-01

    Barnacle muscle cells regulate their volume when exposed to anisotonic conditions. Due to their large size, these cells can be internally perfused. Interestingly, perfused cells maintain their volume regulatory properties (17,21). Thus, the osmotic properties of barnacle muscle cells can be studied under conditions in which the intracellular and extracellular osmolalities, the membrane potential (V(M)), the cell volume and the intracellular pressure can all be measured simultaneously. In this manuscript we report the effect that various rates of isosmotic (1000 mOsm x kg H2O(-1)) intracellular perfusion have on cell volume, intracellular pressure, intracellular osmolality, V(M), and the apparent sarcolemmal hydraulic water permeability (L'p). Replacement of the cytosol with the perfusate at a perfusion rate of 0.83 microl x min(-1) took 120 min. During this transition period, the cell volume increased from 45.1+/-6.9 microl to 73.7+/-5.8 microl, the intracellular osmolality decreased from 1406+/-133 to 1188+/-64 mOsm x kg H2O(-1), and the intracellular pressure underwent a transient drop of 2.8 cm H2O. After 2.5 hr of continuous perfusion at 0.83 microl min(-1), the above mentioned parameters reached steady values: the L'p was 1.35 x 10(-5) cm x sec(-1) x Osm(-1) x kg H2O(-1); cell volume was 67.2+/-6 microl; the intracellular osmolality was 1052+/-10 mOsm x kg H2O(-1); the intracellular pressure was 5.6+/-0.4 cm H2O; V(M) depolarized slowly at a rate of 0.03 mV x min(-1). Stepwise increases in the rate of perfusion (from 0.83 to 3.18 microl min(-1)) produced reversible increases in the intracellular pressure, L'p and cell volume and decreases in intracellular osmolality. We conclude that intracellular perfusion: i/ produces a transient removal of intracellular osmotically active components; ii/ promotes sarcolemmal water filtration; iii/ induces a laminar flow of perfusate at the center of the cell, and iv/ enables calculations of sarcolemmal L'p values under

  3. Conditioned Media from Mesenchymal Stem Cells Enhanced Bone Regeneration in Rat Calvarial Bone Defects

    PubMed Central

    Osugi, Masashi; Yoshimi, Ryoko; Inukai, Takeharu; Hibi, Hideharu; Ueda, Minoru

    2012-01-01

    Tissue engineering has recently become available as a treatment procedure for bone augmentation. However, this procedure has several problems, such as high capital investment and expensive cell culture, complicated safety and quality management issues regarding cell handling, and patient problems with the invasive procedure of cell collection. Moreover, it was reported that stem cells secrete many growth factors and chemokines during their cultivation, which could affect cellular characteristics and behavior. This study investigated the effect of stem-cell-cultured conditioned media on bone regeneration. Cultured conditioned media from human bone marrow–derived mesenchymal stem cells (MSC-CM) enhanced the migration, proliferation, and expression of osteogenic marker genes, such as osteocalcin and Runx2, of rat MSCs (rMSCs) in vitro. MSC-CM includes cytokines such as insulin-like growth factor-1 and vascular endothelial growth factor. In vivo, a prepared bone defect of a rat calvarial model was implanted in five different rat groups using one of the following graft materials: human MSCs/agarose (MSCs), MSC-CM/agarose (MSC-CM), Dulbecco's modified Eagle's medium without serum [DMEM(−)]/agarose [DMEM(−)], PBS/agarose (PBS), and defect only (Defect). After 4 and 8 weeks, implant sections were evaluated using microcomputed tomography (micro-CT) and histological analysis. Micro-CT analysis indicated that the MSC-CM group had a greater area of newly regenerated bone compared with the other groups (p<0.05) and histological analysis at 8 weeks indicated that the newly regenerated bone bridge almost covered the defect. Interestingly, the effects of MSC-CM were stronger than those of the MSC group. In vivo imaging and immunohistochemical staining of transgenic rats expressing green fluorescent protein also showed that migration of rMSCs to the bone defect in the MSC-CM group was greater than in the other groups. These results demonstrated that MSC-CM can regenerate bone

  4. Developing RCM Strategy for Hydrogen Fuel Cells Utilizing On Line E-Condition Monitoring

    NASA Astrophysics Data System (ADS)

    Baglee, D.; Knowles, M. J.

    2012-05-01

    Fuel cell vehicles are considered to be a viable solution to problems such as carbon emissions and fuel shortages for road transport. Proton Exchange Membrane (PEM) Fuel Cells are mainly used in this purpose because they can run at low temperatures and have a simple structure. Yet high maintenance costs and the inherent dangers of maintaining equipment using hydrogen are two main issues which need to be addressed. The development of appropriate and efficient strategies is currently lacking with regard to fuel cell maintenance. A Reliability Centered Maintenance (RCM) approach offers considerable benefit to the management of fuel cell maintenance since it includes an identification and consideration of the impact of critical components. Technological developments in e-maintenance systems, radio-frequency identification (RFID) and personal digital assistants (PDAs) have proven to satisfy the increasing demand for improved reliability, efficiency and safety. RFID technology is used to store and remotely retrieve electronic maintenance data in order to provide instant access to up-to-date, accurate and detailed information. The aim is to support fuel cell maintenance decisions by developing and applying a blend of leading-edge communications and sensor technology including RFID. The purpose of this paper is to review and present the state of the art in fuel cell condition monitoring and maintenance utilizing RCM and RFID technologies. Using an RCM analysis critical components and fault modes are identified. RFID tags are used to store the critical information, possible faults and their cause and effect. The relationship between causes, faults, symptoms and long term implications of fault conditions are summarized. Finally conclusions are drawn regarding suggested maintenance strategies and the optimal structure for an integrated, cost effective condition monitoring and maintenance management system.

  5. Effect of yeast strain and fermentation conditions on the release of cell wall polysaccharides.

    PubMed

    Giovani, Giovanna; Canuti, Valentina; Rosi, Iolanda

    2010-02-28

    To improve our understanding of the factors involved in polysaccharide release during alcoholic fermentation, we investigated three Saccharomyces cerevisiae strains in fermentation trials conducted at two temperatures (25 degrees C and 32 degrees C) and three sugar concentrations (20%, 23.5%, and 27%), with or without supplementation of grape juice with diammonium phosphate (DAP) or microcrystalline cellulose. In two yeast strains, the release of cell wall polysaccharides increased significantly with an increase in fermentation temperature and sugar concentration of the grape juice; the polysaccharide release was greater in stressed conditions, in which the cells were less viable and less metabolically active. In the third strain, the average amount of polysaccharides released into the medium decreased significantly at 32 degrees C with 27% sugar, and increased in grape juice supplemented with DAP. Thus, this strain released more polysaccharides when conditions were nearer to optimal and the yeast cells were more viable and metabolically active. Our results suggest that the yeast strains released cell wall polysaccharides via different mechanisms, and that the cell wall integrity pathway may account for some of the differences in polysaccharide release among the strains.

  6. Nanostructured porous silicon micropatterns as a tool for substrate-conditioned cell research

    PubMed Central

    2012-01-01

    The localized irradiation of Si allows a precise patterning at the microscale of nanostructured materials such as porous silicon (PS). PS patterns with precisely defined geometries can be fabricated using ion stopping masks. The nanoscale textured micropatterns were used to explore their influence as microenvironments for human mesenchymal stem cells (hMSCs). In fact, the change of photoluminescence emission from PS upon aging in physiological solution suggests the intense formation of silanol surface groups, which may play a relevant role in ulterior cell adhesion. The experimental results show that hMSCs are sensitive to the surface micropatterns. In this regard, preliminary β-catenin labeling studies reveal the formation of cell to cell interaction structures, while microtubule orientation is strongly influenced by the selective adhesion conditions. Relevantly, Ki-67 assays support a proliferative state of hMSCs on such nanostructured micropatterns comparable to that of standard cell culture platforms, which reinforce the candidature of porous silicon micropatterns to become a conditioning structure for in vitro culture of HMSCs. PMID:22799489

  7. Tumour cell conditioned medium reveals greater M2 skewing of macrophages in the absence of properdin

    PubMed Central

    Al‐Rayahi, Izzat A.M.; Browning, Michael J.

    2017-01-01

    Abstract Introduction The tumour microenvironment is shaped by the interaction of immune, non immune, and tumour cells present in close proximity. Tumour cells direct the development of a locally immune suppressed state, affecting the activity of anti tumour T cells and preparing the escape phase of tumour development. Macrophages in the tumour typically develop into so‐called tumour associated macrophages with a distinct profile of activities which lead to a reduction in inflammation and antigen presentation. The direct impact of tumour cell conditioned medium on the activity profile of macrophages in dependence of their complement component expression has not yet been investigated. Methods In our in vitro study, macrophages differentiated from bone marrows of properdin deficient and wildtype mice were stimulated with conditioned medium of a syngeneic tumour cell line, B16F10, a mouse melanoma subline. Results In comparison with macrophages from wildtype mice, those from congenic properdin deficient mice showed skewing towards M2 profile, encompassing mRNA expression for genes involved in arginine metabolism, production of type 2 cytokines, and relatively lower surface expression of molecules needed for antigen presentation. Conclusions These data suggest that properdin insufficiency promotes a tumour environment that helps the tumour evade the immune response. PMID:28250926

  8. System for measuring oxygen consumption rates of mammalian cells in static culture under hypoxic conditions.

    PubMed

    Kagawa, Yuki; Miyahara, Hirotaka; Ota, Yuri; Tsuneda, Satoshi

    2016-01-01

    Estimating the oxygen consumption rates (OCRs) of mammalian cells in hypoxic environments is essential for designing and developing a three-dimensional (3-D) cell culture system. However, OCR measurements under hypoxic conditions are infrequently reported in the literature. Here, we developed a system for measuring OCRs at low oxygen levels. The system injects nitrogen gas into the environment and measures the oxygen concentration by an optical oxygen microsensor that consumes no oxygen. The developed system was applied to HepG2 cells in static culture. Specifically, we measured the spatial profiles of the local dissolved oxygen concentration in the medium, then estimated the OCRs of the cells. The OCRs, and also the pericellular oxygen concentrations, decreased nonlinearly as the oxygen partial pressure in the environment decreased from 19% to 1%. The OCRs also depended on the culture period and the matrix used for coating the dish surface. Using this system, we can precisely estimate the OCRs of various cell types under environments that mimic 3-D culture conditions, contributing crucial data for an efficient 3-D culture system design.

  9. Influences of extracellular matrix and of conditioned media on differentiation and invasiveness of trophoblast stem cells.

    PubMed

    Lei, T; Hohn, H-P; Behr, R; Denker, H-W

    2007-01-01

    Embryo implantation in the human and rodents relies on the trophoblast's ability to invade into the uterine stroma, partly depending on proteinases degrading components of basement membrane and underlying extracellular matrix (ECM). We have utilized mouse trophoblast stem (TS) cells (Science, 1998, 282:2072) to study trophoblast invasion and trophoblast-ECM interactions in vitro. On plastic in fibroblast-conditioned medium containing fibroblast growth factor (FGF)-4 and heparin, the cells remain proliferative but display increased differentiation in media without these components. Marker gene expression (Eomes, Pl-1, Tpbp) and invasion assays showed that TS cells exhibit increased invasive capacity when differentiating into giant cells and spongiotrophoblasts in unconditioned media without FGF-4 and heparin. Concomitantly, an up-regulation of matrix metalloproteinases (MMP)-9 and -14 was observed. Culture on gels of the basement membrane-like Matrigel resulted in striking changes in morphology and gene expression. Differentiating TS cells invaded into this ECM in a three-dimensional culture, while in turn ECM contact enhanced differentiation of TS cells and up-regulated the expression of MMP-9 and its tissue inhibitor (TIMP)-3. These findings implicate that the TS cell culture system used in this study can be utilized as a model for studying the regulation of trophoblast-ECM interactions, differentiation, and invasion in vitro.

  10. Electrical conditioning of adipose-derived stem cells in a multi-chamber culture platform.

    PubMed

    Pavesi, A; Soncini, M; Zamperone, A; Pietronave, S; Medico, E; Redaelli, A; Prat, M; Fiore, G B

    2014-07-01

    In tissue engineering, several factors play key roles in providing adequate stimuli for cells differentiation, in particular biochemical and physical stimuli, which try to mimic the physiological microenvironments. Since electrical stimuli are important in the developing heart, we have developed an easy-to-use, cost-effective cell culture platform, able to provide controlled electrical stimulation aimed at investigating the influence of the electric field in the stem cell differentiation process. This bioreactor consists of an electrical stimulator and 12 independent, petri-like culture chambers and a 3-D computational model was used to characterize the distribution and the intensity of the electric field generated in the cell culture volume. We explored the effects of monophasic and biphasic square wave pulse stimulation on a mouse adipose-derived stem cell line (m17.ASC) comparing cell viability, proliferation, protein, and gene expression. Both monophasic (8 V, 2 ms, 1 Hz) and biphasic (+4 V, 1 ms and -4 V, 1 ms; 1 Hz) stimulation were compatible with cell survival and proliferation. Biphasic stimulation induced the expression of Connexin 43, which was found to localize also at the cell membrane, which is its recognized functional mediating intercellular electrical coupling. Electrically stimulated cells showed an induced transcriptional profile more closely related to that of neonatal cadiomyocytes, particularly for biphasic stimulation. The developed platform thus allowed to set-up precise conditions to drive adult stem cells toward a myocardial phenotype solely by physical stimuli, in the absence of exogenously added expensive bioactive molecules, and can thus represent a valuable tool for translational applications for heart tissue engineering and regeneration.

  11. Electromechanical Conditioning of Adult Progenitor Cells Improves Recovery of Cardiac Function After Myocardial Infarction.

    PubMed

    Llucià-Valldeperas, Aida; Soler-Botija, Carolina; Gálvez-Montón, Carolina; Roura, Santiago; Prat-Vidal, Cristina; Perea-Gil, Isaac; Sanchez, Benjamin; Bragos, Ramon; Vunjak-Novakovic, Gordana; Bayes-Genis, Antoni

    2017-03-01

    Cardiac cells are subjected to mechanical and electrical forces, which regulate gene expression and cellular function. Therefore, in vitro electromechanical stimuli could benefit further integration of therapeutic cells into the myocardium. Our goals were (a) to study the viability of a tissue-engineered construct with cardiac adipose tissue-derived progenitor cells (cardiac ATDPCs) and (b) to examine the effect of electromechanically stimulated cardiac ATDPCs within a myocardial infarction (MI) model in mice for the first time. Cardiac ATDPCs were electromechanically stimulated at 2-millisecond pulses of 50 mV/cm at 1 Hz and 10% stretching during 7 days. The cells were harvested, labeled, embedded in a fibrin hydrogel, and implanted over the infarcted area of the murine heart. A total of 39 animals were randomly distributed and sacrificed at 21 days: groups of grafts without cells and with stimulated or nonstimulated cells. Echocardiography and gene and protein analyses were also carried out. Physiologically stimulated ATDPCs showed increased expression of cardiac transcription factors, structural genes, and calcium handling genes. At 21 days after implantation, cardiac function (measured as left ventricle ejection fraction between presacrifice and post-MI) increased up to 12% in stimulated grafts relative to nontreated animals. Vascularization and integration with the host blood supply of grafts with stimulated cells resulted in increased vessel density in the infarct border region. Trained cells within the implanted fibrin patch expressed main cardiac markers and migrated into the underlying ischemic myocardium. To conclude, synchronous electromechanical cell conditioning before delivery may be a preferred alternative when considering strategies for heart repair after myocardial infarction. Stem Cells Translational Medicine 2017;6:970-981.

  12. Establishment and characterization of a new conditionally immortalized human astrocyte cell line.

    PubMed

    Furihata, Tomomi; Ito, Ryo; Kamiichi, Atsuko; Saito, Kosuke; Chiba, Kan

    2016-01-01

    Astrocytes are the most abundant cell types in mammalian brains, within which they participate in various neuronal activities, partly by utilizing the numerous transporters expressed at their plasma membranes. Accordingly, detailed characterization of astrocytic functions, including transporters, are essential for understanding of mechanistic basis of normal brain functions, as well as the pathogenesis and treatment of various brain diseases. As a part of overall efforts to facilitate such studies, this study reports on the establishment of a new human astrocyte cell line, which is hereafter referred to as human astrocyte/conditionally immortalized, clone 35 (HASTR/ci35). This line, which was developed utilizing a cell immortalization method, showed excellent proliferative ability and expressed various astrocyte markers, including glial fibrillary acidic protein. When co-cultured with neuronal cells, HASTR/ci35 cells could facilitate their dendritic network formation. Furthermore, HASTR/ci35 cells not only possessed significant glutamate and adenosine transporter activities but also exhibited organic ion transporter activities. To summarize, HASTR/ci35 cells possess several key astrocytic characteristics, including various transporter functions, while simultaneously showing infinite proliferation and scalability. Based on these findings, HASTR/ci35 cells can be expected to contribute significantly to various human astrocyte study fields. In vitro astrocyte models are valuable experimental tools in various astrocyte studies. Here, we report the establishment of a new human astrocyte cell line, HASTR/ci35, which show various key astrocyte properties, including astrocytic transporter activities, glycogen storage and facilitation of neuronal cell differentiation. Thus, HASTR/ci35 is expected to significantly contribute to advances toward detailed understanding of human astrocyte functions.

  13. Osteocalcin protects pancreatic beta cell function and survival under high glucose conditions

    SciTech Connect

    Kover, Karen; Yan, Yun; Tong, Pei Ying; Watkins, Dara; Li, Xiaoyu; Tasch, James; Hager, Melissa; Clements, Mark; Moore, Wayne V.

    2015-06-19

    Diabetes is characterized by progressive beta cell dysfunction and loss due in part to oxidative stress that occurs from gluco/lipotoxicity. Treatments that directly protect beta cell function and survival in the diabetic milieu are of particular interest. A growing body of evidence suggests that osteocalcin, an abundant non-collagenous protein of bone, supports beta cell function and proliferation. Based on previous gene expression data by microarray, we hypothesized that osteocalcin protects beta cells from glucose-induced oxidative stress. To test our hypothesis we cultured isolated rat islets and INS-1E cells in the presence of normal, high, or high glucose ± osteocalcin for up to 72 h. Oxidative stress and viability/mitochondrial function were measured by H{sub 2}O{sub 2} assay and Alamar Blue assay, respectively. Caspase 3/7 activity was also measured as a marker of apoptosis. A functional test, glucose stimulated insulin release, was conducted and expression of genes/protein was measured by qRT-PCR/western blot/ELISA. Osteocalcin treatment significantly reduced high glucose-induced H{sub 2}O{sub 2} levels while maintaining viability/mitochondrial function. Osteocalcin also significantly improved glucose stimulated insulin secretion and insulin content in rat islets after 48 h of high glucose exposure compared to untreated islets. As expected sustained high glucose down-regulated gene/protein expression of INS1 and BCL2 while increasing TXNIP expression. Interestingly, osteocalcin treatment reversed the effects of high glucose on gene/protein expression. We conclude that osteocalcin can protect beta cells from the negative effects of glucose-induced oxidative stress, in part, by reducing TXNIP expression, thereby preserving beta cell function and survival. - Highlights: • Osteocalcin reduces glucose-induced oxidative stress in beta cells. • Osteocalcin preserves beta cell function and survival under stress conditions. • Osteocalcin reduces glucose

  14. Donor Chimerism Early after Reduced-intensity Conditioning Hematopoietic Stem Cell Transplantation Predicts Relapse and Survival

    PubMed Central

    Koreth, John; Kim, Haesook T.; Nikiforow, Sarah; Milford, Edgar L.; Armand, Philippe; Cutler, Corey; Glotzbecker, Brett; Ho, Vincent T.; Antin, Joseph H.; Soiffer, Robert J.; Ritz, Jerome; Alyea, Edwin P.

    2015-01-01

    The impact of early donor cell chimerism on outcomes of T-replete reduced-intensity conditioning (RIC) hematopoietic stem cell transplantation (HSCT) is ill-defined. We evaluated day 30 (D30) and 100 (D100) total donor cell chimerism after RIC HSCT undertaken between 2002 and 2010 at our institution, excluding patients who died or relapsed before D30. When available, donor T-cell chimerism was also assessed. The primary outcome was overall survival (OS). Secondary outcomes included progression-free survival (PFS), relapse and non-relapse mortality (NRM). 688 patients with hematologic malignancies (48% myeloid; 52% lymphoid) and a median age of 57 years (range, 18-74) undergoing RIC HSCT with T-replete donor grafts (97% peripheral blood; 92% HLA-matched) and median follow-up of 58.2 months (range, 12.6-120.7) were evaluated. In multivariable analysis total donor cell and T-cell chimerism at D30 and D100 each predicted RIC HSCT outcomes, with D100 total donor cell chimerism most predictive. D100 total donor cell chimerism <90% was associated with increased relapse (HR 2.54, 95% CI 1.83-3.51, p<0.0001), impaired PFS (HR 2.01, 95% CI 1.53-2.65, p<0.0001) and worse OS (1.50, 95% CI 1.11-2.04, p=0.009), but not NRM (HR 0.76; 95% CI 0.44-2.27, p=0.33). There was no additional utility of incorporating sustained D30-D100 total donor cell chimerism, or T-cell chimerism. Low donor chimerism early after RIC HSCT is an independent risk factor for relapse and impaired survival. Donor chimerism assessment early after RIC HSCT can prognosticate for long-term outcomes and help identify high-risk patient cohorts that may benefit from additional therapeutic interventions. PMID:24907627

  15. Mechanisms of diabetic neuropathy: Schwann cells.

    PubMed

    Mizisin, Andrew P

    2014-01-01

    As ensheathing and secretory cells, Schwann cells are a ubiquitous and vital component of the endoneurial microenvironment of peripheral nerves. The interdependence of axons and their ensheathing Schwann cells predisposes each to the impact of injury in the other. Further, the dependence of the blood-nerve interface on trophic support from Schwann cells during development, adulthood, and after injury suggests these glial cells promote the structural and functional integrity of nerve trunks. Here, the developmental origin, injury-induced changes, and mature myelinating and nonmyelinating phenotypes of Schwann cells are reviewed prior to a description of nerve fiber pathology and consideration of pathogenic mechanisms in human and experimental diabetic neuropathy. A fundamental role for aldose-reductase-containing Schwann cells in the pathogenesis of diabetic neuropathy, as well as the interrelationship of pathogenic mechanisms, is indicated by the sensitivity of hyperglycemia-induced biochemical alterations, such as polyol pathway flux, formation of reactive oxygen species, generation of advanced glycosylation end products (AGEs) and deficient neurotrophic support, to blocking polyol pathway flux.

  16. Evaluation of ultralow platinum loaded electrodes in PEM fuel cell at ambient conditions

    SciTech Connect

    Swathirajan, S.; Mikhail, Y.M.

    1994-12-31

    A Nafion{reg_sign} slurry coating method was used to prepare thin film electrode-membrane assemblies with ultralow platinum loadings in the range 0.025--0.09 mg/cm{sup 2} for a proton exchange membrane (PEM) fuel cell. This assembly, evaluated at ambient conditions, showed a catalyst utilization that was considerably higher by a factor of 7 when compared to an assembly prepared from the commercially available E-tek electrodes. A model for the factors determining catalyst utilization in fuel cell electrodes, and the optimization of the thin electrode composition are described.

  17. Treosulfan-based conditioning regimen for allogeneic haematopoietic stem cell transplantation in children with sickle cell disease.

    PubMed

    Strocchio, Luisa; Zecca, Marco; Comoli, Patrizia; Mina, Tommaso; Giorgiani, Giovanna; Giraldi, Eugenia; Vinti, Luciana; Merli, Pietro; Regazzi, Mario; Locatelli, Franco

    2015-06-01

    Although allogeneic haematopoietic stem cell transplantation (HSCT) still represents the only consolidated possibility of cure for sickle cell disease (SCD) patients, its use has been limited by the risk of morbidity and mortality associated with conventional myeloablative therapy. The introduction of treosulfan to replace busulfan in conditioning regimens has recently been explored by virtue of its lower toxicity profile. We report our experience with a treosulfan/thiotepa/fludarabine conditioning for human leucocyte antigen (HLA)-matched sibling or unrelated donor-HSCT in 15 children with SCD, and compare patient outcomes with those of a historical cohort (15 patients) given a busulfan-based regimen. Engraftment was achieved in 28 out of 30 patients (93%), with one case of graft failure in either group. The conditioning regimen was well tolerated in both groups, with no cases of grade III-IV regimen-related toxicity. The 7-year overall survival (OS) and disease-free survival (DFS) for the whole cohort were 100% and 93%, respectively, with a 93% DFS in both busulfan and treosulfan groups. No SCD-related adverse events occurred after engraftment in patients with complete or mixed donor chimerism. This retrospective analysis suggests that a treosulfan-based conditioning regimen is able to ensure engraftment with excellent OS/DFS and low regimen-related toxicity in patients with SCD.

  18. Comparison of mesenchymal stem cells derived from gingival tissue and periodontal ligament in different incubation conditions.

    PubMed

    Yang, Hao; Gao, Li-Na; An, Ying; Hu, Cheng-Hu; Jin, Fang; Zhou, Jun; Jin, Yan; Chen, Fa-Ming

    2013-09-01

    Gingival tissue-derived mesenchymal stem cells (MSCs) were recently identified and characterized as having multipotential differentiation and immunomodulatory properties in vitro and in vivo, and they represent new postnatal stem cell types for cytotherapy and regenerative medicine. However, the utility of gingival MSCs (GMSCs) as alternatives to periodontal ligament stem cells (PDLSCs), which have been demonstrated to be effective but with limited cell availability and reduced clinical feasibility, for periodontal regeneration in a previously diseased/inflamed environment remains obscure. In this study, patient-matched human GMSCs and PDLSCs were evaluated in terms of their colony-forming ability, proliferative capacity, cell surface epitopes, multi-lineage differentiation potentials, and related gene expression when incubated in different designed culture conditions, with or without the presence of inflammatory cytokines. An in vivo ectopic transplantation model using transplants from inflammatory cytokine-treated or untreated cells was applied to assess bone formation. We found that cells derived from both tissues expressed MSC markers, including CD146, CD105, CD90, CD29, and STRO-1. Both cells successfully differentiated under osteogenic, adipogenic, and chondrogenic microenvironments; PDLSCs displayed a more effective differentiation potential in all of the incubation conditions compared to GMSCs (P < 0.01). Although inflammatory cytokine-treated GMSCs and PDLSCs are inferior to normally cultured, patient and tissue-matched cells in terms of their osteogenic capacity and regenerative potential (P < 0.05), they retain the capacity for osteoblastic and adipose differentiation, as well as ectopic bone formation, similar to what has been demonstrated for other MSCs. Interestingly, GMSCs exhibited fewer inflammation-related changes in terms of osteogenic potential in vitro and bone formation in vivo compared to PDLSCs (P < 0.01). These results suggest

  19. Transparent Boundary Condition for Oseen-Frank Model. Application for NLC Cells With Patterned Electrodes

    NASA Astrophysics Data System (ADS)

    Orquín-Serrano, I.; Vijande, J.; Villatoro, F. R.; Ferrando, A.; Fernández de Córdoba, P.; Michinel, H.

    2015-04-01

    In the present work a novel application of Transparent Boundary Conditions (TBC) to nematic liquid crystal cells (NLCC) with planar alignment and a patterned electrode is studied. This device is attracting great interest since it allows soliton steering by optically and externally induced waveguides. We employ the continuum Oseen-Frank theory to find the tilt and twist angle distributions in the cell under the one-constant approximation. The electric field distribution takes into account the whole 2D permittivity tensor for the transverse coordinates. Standard finite difference time domain methods together with an iterative method is applied to find an approximate solution to our coupled problem. A novel class of TBC is used to correctly define the boundary for both the distortion angle problem and the electric field distribution when using patterned electrodes. Thus, we achieve an important decrease of computational needs when solving this kind of problems and we are also capable of exploring weak anchoring conditions for NLCC.

  20. IL-10 conditioning of human skin affects the distribution of migratory dendritic cell subsets and functional T cell differentiation.

    PubMed

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

    2013-01-01

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

  1. Impact of NKT Cells and LFA-1 on Liver Regeneration under Subseptic Conditions

    PubMed Central

    Fehlner, Karin; Weisser, Tanja; Cheng, Zhangjun; Lu, Miao; Höchst, Bastian; Bolzer, Andreas; Wang, Baocai; Hartmann, Daniel; Assfalg, Volker; Sunami, Yoshiaki; Schlitter, Anna Melissa; Friess, Helmut; Hüser, Norbert; Laschinger, Melanie

    2016-01-01

    Background Activation of the immune system in terms of subseptic conditions during liver regeneration is of paramount clinical importance. However, little is known about molecular mechanisms and their mediators that control hepatocyte proliferation. We sought to determine the functional role of immune cells, especially NKT cells, in response to partial hepatectomy (PH), and to uncover the impact of the integrin lymphocyte function-associated antigen-1 (LFA-1) on liver regeneration in a subseptic setting. Methods Wild-type (WT) and LFA-1-/- mice underwent a 2/3 PH and low-dose lipopolysaccharid (LPS) application. Hepatocyte proliferation, immune cell infiltration, and cytokine profile in the liver parenchyma were determined. Results Low-dose LPS application after PH results in a significant delay of liver regeneration between 48h and 72h, which is associated with a reduced number of CD3+ cells within the regenerating liver. In absence of LFA-1, an impaired regenerative capacity was observed under low-dose LPS application. Analysis of different leukocyte subpopulations showed less CD3+NK1.1+ NKT cells in the liver parenchyma of LFA-1-/- mice after PH and LPS application compared to WT controls, while CD3-NK1.1+ NK cells markedly increased. Concordantly with this observation, lower levels of NKT cell related cytokines IL-12 and IL-23 were expressed in the regenerating liver of LFA-1-/- mice, while the expression of NK cell-associated CCL5 and IL-10 was increased compared to WT mice. Conclusion A subseptic situation negatively alters hepatocyte proliferation. Within this scenario, we suggest an important impact of NKT cells and postulate a critical function for LFA-1 during processes of liver regeneration. PMID:27977747

  2. Corrosion behavior of iron and nickel base alloys under solid oxide fuel cell exposure conditions

    SciTech Connect

    Ziomek-Moroz, M.; Holcomb, G.R.; Covino, B.S., Jr.; Bullard, S.J.

    2006-03-01

    Topography and phase composition of the scales formed on commercial ferritic stainless steels and experimental low CTE nickel-based alloys were studied in atmospheres simulating solid oxide fuel cell (SOFC) environments. The materials were studied under dual environment conditions with air on one side of the sample and carbon monoxide on the other side at 750°C. Surface characterization techniques, such as scanning electron microscopy and X-ray diffraction analysis were used in this study.

  3. Visualization of Fuel Cell Water Transport and Performance Characterization under Freezing Conditions

    SciTech Connect

    Kandlikar, Satish G.; Lu, Zijie; Rao, Navalgund; Sergi, Jacqueline; Rath, Cody; McDade, Christopher; Trabold, Thomas; Owejan, Jon; Gagliardo, Jeffrey; Allen, Jeffrey; Yassar, Reza S.; Medici, Ezequiel; Herescu, Alexandru

    2010-05-30

    In this program, Rochester Institute of Technology (RIT), General Motors (GM) and Michigan Technological University (MTU) have focused on fundamental studies that address water transport, accumulation and mitigation processes in the gas diffusion layer and flow field channels of the bipolar plate. These studies have been conducted with a particular emphasis on understanding the key transport phenomena which control fuel cell operation under freezing conditions.

  4. GBT440 Inhibits Sickling of Sickle Cell Trait Blood Under In Vitro Conditions Mimicking Strenuous Exercise

    PubMed Central

    Dufu, Kobina; Lehrer-Graiwer, Josh; Ramos, Eleanor; Oksenberg, Donna

    2016-01-01

    In sickle cell trait (SCT), hemoglobin A (HbA) and S (HbS) are co-expressed in each red blood cell (RBC). While homozygous expression of HbS (HbSS) leads to polymerization and sickling of RBCs resulting in sickle cell disease (SCD) characterized by hemolytic anemia, painful vaso-occlusive episodes and shortened life-span, SCT is considered a benign condition usually with minor or no complications related to sickling. However, physical activities that cause increased tissue oxygen demand, dehydration and/or metabolic acidosis leads to increased HbS polymerization and life-threatening complications including death. We report that GBT440, an agent being developed for the treatment of SCD, increases the affinity of oxygen for Hb and inhibits in vitro polymerization of a mixture of HbS and HbA that simulates SCT blood. Moreover, GBT440 prevents sickling of SCT blood under in vitro conditions mimicking strenuous exercise with hypoxia, dehydration and acidosis. Together, our results indicate that GBT440 may have the potential to protect SCT individuals from sickling-related complications during conditions that favor HbS polymerization. PMID:27757216

  5. Lent-On-Plus Lentiviral vectors for conditional expression in human stem cells.

    PubMed

    Benabdellah, Karim; Muñoz, Pilar; Cobo, Marién; Gutierrez-Guerrero, Alejandra; Sánchez-Hernández, Sabina; Garcia-Perez, Angélica; Anderson, Per; Carrillo-Gálvez, Ana Belén; Toscano, Miguel G; Martin, Francisco

    2016-11-17

    Conditional transgene expression in human stem cells has been difficult to achieve due to the low efficiency of existing delivery methods, the strong silencing of the transgenes and the toxicity of the regulators. Most of the existing technologies are based on stem cells clones expressing appropriate levels of tTA or rtTA transactivators (based on the TetR-VP16 chimeras). In the present study, we aim the generation of Tet-On all-in-one lentiviral vectors (LVs) that tightly regulate transgene expression in human stem cells using the original TetR repressor. By using appropriate promoter combinations and shielding the LVs with the Is2 insulator, we have constructed the Lent-On-Plus Tet-On system that achieved efficient transgene regulation in human multipotent and pluripotent stem cells. The generation of inducible stem cell lines with the Lent-ON-Plus LVs did not require selection or cloning, and transgene regulation was maintained after long-term cultured and upon differentiation toward different lineages. To our knowledge, Lent-On-Plus is the first all-in-one vector system that tightly regulates transgene expression in bulk populations of human pluripotent stem cells and its progeny.

  6. Lent-On-Plus Lentiviral vectors for conditional expression in human stem cells

    PubMed Central

    Benabdellah, Karim; Muñoz, Pilar; Cobo, Marién; Gutierrez-Guerrero, Alejandra; Sánchez-Hernández, Sabina; Garcia-Perez, Angélica; Anderson, Per; Carrillo-Gálvez, Ana Belén; Toscano, Miguel G.; Martin, Francisco

    2016-01-01

    Conditional transgene expression in human stem cells has been difficult to achieve due to the low efficiency of existing delivery methods, the strong silencing of the transgenes and the toxicity of the regulators. Most of the existing technologies are based on stem cells clones expressing appropriate levels of tTA or rtTA transactivators (based on the TetR-VP16 chimeras). In the present study, we aim the generation of Tet-On all-in-one lentiviral vectors (LVs) that tightly regulate transgene expression in human stem cells using the original TetR repressor. By using appropriate promoter combinations and shielding the LVs with the Is2 insulator, we have constructed the Lent-On-Plus Tet-On system that achieved efficient transgene regulation in human multipotent and pluripotent stem cells. The generation of inducible stem cell lines with the Lent-ON-Plus LVs did not require selection or cloning, and transgene regulation was maintained after long-term cultured and upon differentiation toward different lineages. To our knowledge, Lent-On-Plus is the first all-in-one vector system that tightly regulates transgene expression in bulk populations of human pluripotent stem cells and its progeny. PMID:27853296

  7. Carnitine palmitoyltransferase 1C promotes cell survival and tumor growth under conditions of metabolic stress

    PubMed Central

    Zaugg, Kathrin; Yao, Yi; Reilly, Patrick T.; Kannan, Karuppiah; Kiarash, Reza; Mason, Jacqueline; Huang, Ping; Sawyer, Suzanne K.; Fuerth, Benjamin; Faubert, Brandon; Kalliomäki, Tuula; Elia, Andrew; Luo, Xunyi; Nadeem, Vincent; Bungard, David; Yalavarthi, Sireesha; Growney, Joseph D.; Wakeham, Andrew; Moolani, Yasmin; Silvester, Jennifer; Ten, Annick You; Bakker, Walbert; Tsuchihara, Katsuya; Berger, Shelley L.; Hill, Richard P.; Jones, Russell G.; Tsao, Ming; Robinson, Murray O.; Thompson, Craig B.; Pan, Guohua; Mak, Tak W.

    2011-01-01

    Tumor cells gain a survival/growth advantage by adapting their metabolism to respond to environmental stress, a process known as metabolic transformation. The best-known aspect of metabolic transformation is the Warburg effect, whereby cancer cells up-regulate glycolysis under aerobic conditions. However, other mechanisms mediating metabolic transformation remain undefined. Here we report that carnitine palmitoyltransferase 1C (CPT1C), a brain-specific metabolic enzyme, may participate in metabolic transformation. CPT1C expression correlates inversely with mammalian target of rapamycin (mTOR) pathway activation, contributes to rapamycin resistance in murine primary tumors, and is frequently up-regulated in human lung tumors. Tumor cells constitutively expressing CPT1C show increased fatty acid (FA) oxidation, ATP production, and resistance to glucose deprivation or hypoxia. Conversely, cancer cells lacking CPT1C produce less ATP and are more sensitive to metabolic stress. CPT1C depletion via siRNA suppresses xenograft tumor growth and metformin responsiveness in vivo. CPT1C can be induced by hypoxia or glucose deprivation and is regulated by AMPKα. Cpt1c-deficient murine embryonic stem (ES) cells show sensitivity to hypoxia and glucose deprivation and altered FA homeostasis. Our results indicate that cells can use a novel mechanism involving CPT1C and FA metabolism to protect against metabolic stress. CPT1C may thus be a new therapeutic target for the treatment of hypoxic tumors. PMID:21576264

  8. Fracture process of nonstoichiometric oxide based solid oxide fuel cell under oxidizing/reducing gradient conditions

    NASA Astrophysics Data System (ADS)

    Sato, Kazuhisa; Yashiro, Keiji; Kawada, Tatsuya; Yugami, Hiroo; Hashida, Toshiyuki; Mizusaki, Junichiro

    The influence of chemically induced expansion on the fracture damage of a nonstoichiometric oxide (ceria) based solid oxide fuel cell (SOFC) single cell laminate was investigated by using numerical stress analyses under oxidizing/reducing gradient condition. The single cell examined in this study was composed of electrolyte (Ce 0.8Sm 0.2O 2- δ), anode (Cermets of Ni-Ce 0.8Sm 0.2O 2- δ), and cathode (La 0.6Sr 0.4Co 0.2Fe 0.8O 3- δ), respectively. The finite element method (FEM) was employed to calculate the residual stress, thermal stresses, and chemically induced expansion stresses for the single cell. The residual and thermal stresses were calculated much smaller than the fracture strength of the individual components of the single cell. On the other hand, the chemically induced expansion stresses were shown to remarkably increase for the temperature range greater than 973 K and accounted their magnitude for primary part of the induced stress. It was shown from the FEM that the maximum circumferential stress induced in the single cell exceeded the fracture strength of the individual components at the onset of the fracture damage detect by acoustic emission (AE) method.

  9. Detection and manipulation of live antigen-expressing cells using conditionally stable nanobodies

    PubMed Central

    Tang, Jonathan CY; Drokhlyansky, Eugene; Etemad, Behzad; Rudolph, Stephanie; Guo, Binggege; Wang, Sui; Ellis, Emily G; Li, Jonathan Z; Cepko, Constance L

    2016-01-01

    The ability to detect and/or manipulate specific cell populations based upon the presence of intracellular protein epitopes would enable many types of studies and applications. Protein binders such as nanobodies (Nbs) can target untagged proteins (antigens) in the intracellular environment. However, genetically expressed protein binders are stable regardless of antigen expression, complicating their use for applications that require cell-specificity. Here, we created a conditional system in which the stability of an Nb depends upon an antigen of interest. We identified Nb framework mutations that can be used to rapidly create destabilized Nbs. Fusion of destabilized Nbs to various proteins enabled applications in living cells, such as optogenetic control of neural activity in specific cell types in the mouse brain, and detection of HIV-infected human cells by flow cytometry. These approaches are generalizable to other protein binders, and enable the rapid generation of single-polypeptide sensors and effectors active in cells expressing specific intracellular epitopes. DOI: http://dx.doi.org/10.7554/eLife.15312.001 PMID:27205882

  10. Effects of Low-dose Triamcinolone Acetonide on Rat Retinal Progenitor Cells under Hypoxia Condition

    PubMed Central

    Xing, Yao; Cui, Li-Jun; Kang, Qian-Yan

    2016-01-01

    Background: Retinal degenerative diseases are the leading causes of blindness in developed world. Retinal progenitor cells (RPCs) play a key role in retina restoration. Triamcinolone acetonide (TA) is widely used for the treatment of retinal degenerative diseases. In this study, we investigated the role of TA on RPCs in hypoxia condition. Methods: RPCs were primary cultured and identified by immunofluorescence staining. Cells were cultured under normoxia, hypoxia 6 h, and hypoxia 6 h with TA treatment conditions. For the TA treatment groups, after being cultured under hypoxia condition for 6 h, RPCs were treated with different concentrations of TA for 48–72 h. Cell viability was measured by cell counting kit-8 (CCK-8) assay. Cell cycle was detected by flow cytometry. Western blotting was employed to examine the expression of cyclin D1, Akt, p-Akt, nuclear factor (NF)-κB p65, and caspase-3. Results: CCK-8 assays indicated that the viability of RPCs treated with 0.01 mg/ml TA in hypoxia group was improved after 48 h, comparing with control group (P < 0.05). After 72 h, the cell viability was enhanced in both 0.01 mg/ml and 0.02 mg/ml TA groups compared with control group (all P < 0.05). Flow cytometry revealed that there were more cells in S-phase in hypoxia 6 h group than in normoxia control group (P < 0.05). RPCs in S and G2/M phases decreased in groups given TA, comparing with other groups (all P < 0.05). There was no significant difference in the total Akt protein expression among different groups, whereas upregulation of p-Akt and NF-κB p65 protein expression and downregulation of caspase-3 and cyclin D1 protein expression were observed in 0.01 mg/ml TA group, comparing with hypoxia 6 h group and control group (all P < 0.05). Conclusion: Low-dose TA has anti-apoptosis effect on RPCs while it has no stimulatory effect on cell proliferation. PMID:27364798

  11. Dissociation and Re-Aggregation of Multicell-Ensheathed Fragments Responsible for Rapid Production of Massive Clumps of Leptothrix Sheaths.

    PubMed

    Kunoh, Tatsuki; Nagaoka, Noriyuki; McFarlane, Ian R; Tamura, Katsunori; El-Naggar, Mohamed Y; Kunoh, Hitoshi; Takada, Jun

    2016-08-01

    Species of the Fe/Mn-oxidizing bacteria Leptothrix produce tremendous amounts of microtubular, Fe/Mn-encrusted sheaths within a few days in outwells of groundwater that can rapidly clog water systems. To understand this mode of rapid sheath production and define the timescales involved, behaviors of sheath-forming Leptothrix sp. strain OUMS1 were examined using time-lapse video at the initial stage of sheath formation. OUMS1 formed clumps of tangled sheaths. Electron microscopy confirmed the presence of a thin layer of bacterial exopolymer fibrils around catenulate cells (corresponding to the immature sheath). In time-lapse videos, numerous sheath filaments that extended from the periphery of sheath clumps repeatedly fragmented at the apex of the same fragment, the fragments then aggregated and again elongated, eventually forming a large sheath clump comprising tangled sheaths within two days. In this study, we found that fast microscopic fragmentation, dissociation, re-aggregation and re-elongation events are the basis of the rapid, massive production of Leptothrix sheaths typically observed at macroscopic scales.

  12. Dissociation and Re-Aggregation of Multicell-Ensheathed Fragments Responsible for Rapid Production of Massive Clumps of Leptothrix Sheaths

    PubMed Central

    Kunoh, Tatsuki; Nagaoka, Noriyuki; McFarlane, Ian R.; Tamura, Katsunori; El-Naggar, Mohamed Y.; Kunoh, Hitoshi; Takada, Jun

    2016-01-01

    Species of the Fe/Mn-oxidizing bacteria Leptothrix produce tremendous amounts of microtubular, Fe/Mn-encrusted sheaths within a few days in outwells of groundwater that can rapidly clog water systems. To understand this mode of rapid sheath production and define the timescales involved, behaviors of sheath-forming Leptothrix sp. strain OUMS1 were examined using time-lapse video at the initial stage of sheath formation. OUMS1 formed clumps of tangled sheaths. Electron microscopy confirmed the presence of a thin layer of bacterial exopolymer fibrils around catenulate cells (corresponding to the immature sheath). In time-lapse videos, numerous sheath filaments that extended from the periphery of sheath clumps repeatedly fragmented at the apex of the same fragment, the fragments then aggregated and again elongated, eventually forming a large sheath clump comprising tangled sheaths within two days. In this study, we found that fast microscopic fragmentation, dissociation, re-aggregation and re-elongation events are the basis of the rapid, massive production of Leptothrix sheaths typically observed at macroscopic scales. PMID:27490579

  13. Changes in functional activity of bone tissue cells under space flight conditions.

    NASA Astrophysics Data System (ADS)

    Rodionova, Natalia; Nesterenko, Olga; Kabitskaya, Olga

    The space flight conditions affect considerably the state of bone tissue, leading to the development of osteoporosis and osteopenia. Many aspects of reactions of bone tissue cells still remain unclear until now. With the use of electron microscopy we studied the samples gathered from the femoral bonеs metaphyses of rats flown on board the space laboratory (Spacelab - 2) during 2 weeks and samples from tibial bones of mice C57 Black ( Bion М-1). It was established, that under microgravity conditions there occur remodelling processes in a spongy bone related with a deficit of support load. In this work the main attention is focused on studying the ultrastructure of osteogenetic cells and osteoclasts. The degree of differentiation and functional state are evaluated according to the degree of development of organelles for specific biosynthesis: rough endoplasmic reticulum (RER), Golgy complex (GC), as well as the state of mitochondria and cell nucleus. As compared with a synchronous control, the population of osteogenetic cells from zones of bone reconstruction shows a decrease in the number of functionally active forms. We can judge of this from the reduction of a specific volume of RER, GC, mitochondria in osteoblasts. RER loses architectonics typical for osteoblasts and, as against the control, is represented by short narrow canaliculi distributed throughout the cytoplasm; some canals disintegrate. GC is slightly pronounced, mitochondria become smaller in size and acquire an optically dark matrix. These phenomena are supposed to be associated with the desorganization of microtubules and microfilaments in the cells under microgravity conditions. The population of osteogenetic cells shows a decrease in the number of differentiating osteoblasts and an increase in the number of little-differentiated stromal cells. In the population of osteoblasts, degrading and apoptotic cells are sometimes encountered. Such zones show a numerical increase of monocytic cells and

  14. Modeling hydrogen starvation conditions in proton-exchange membrane fuel cells

    NASA Astrophysics Data System (ADS)

    Ohs, Jan Hendrik; Sauter, Ulrich; Maass, Sebastian; Stolten, Detlef

    In this study, a steady state and isothermal 2D-PEM fuel cell model is presented. By simulation of a single cell along the channel and in through-plane direction, its behaviour under hydrogen starvation due to nitrogen dilution is analysed. Under these conditions, carbon corrosion and water electrolysis are observed on the cathode side. This phenomenon, causing severe cell degradation, is known as reverse current decay mechanism in literature. Butler-Volmer equations are used to model the electrochemical reactions. In addition, we account for permeation of gases through the membrane and for the local water content within the membrane. The results show that the membrane potential locally drops in areas starved from hydrogen. This leads to potential gradients >1.2 V between electrode and membrane on the cathode side resulting in significant carbon corrosion and electrolysis reaction rates. The model enables the analysis of sub-stoichiometric states occurring during anode gas recirculation or load transients.

  15. Focal contacts organization in osteoblastic cells under microgravity and cyclic deformation conditions

    NASA Astrophysics Data System (ADS)

    Guignandon, Alain; Akhouayri, Omar; Laroche, Norbert; Lafage-Proust, Marie-Hélène; Alexandre, Christian; Vico, Laurence

    2003-10-01

    We compared quantitatively vinculin-related adhesion parameters in osteoblastic cells submitted to opposite mechanical stresses, i.e., low deformation and frequency strain regimens (stretch condition) and microgravity exposure (relaxed condition). Cyclic deformation induced a biphasic response comprising new focal contacts formation followed by their clustering in ROS cells. Microgravity exposure induced a reduction in focal contact number and clustering in ROS cells. We previously demonstrated that 1% cyclic deformations at 0.05 Hz during a daily 10 min episode over 7 days stimulated ROS 17/2.8 growth as compared to static culture whereas relaxed ROS proliferated similarly to static culture (BC). To evaluate whether the proliferation (stretch) or the survival (relaxed) status of ROS cells influences focal contact organization, we inhibited ERKs proliferative-dependent pathway. Inhibition of proliferation by PD98059 was overcome although not fully restored by stretch. Furthermore stretch-induced clustering of vinculin-positive contacts still occurs in the presence of ERKs inhibitor, whereas the increase in focal contact number is abolished. In conclusion, we showed that focal contacts are mechanoeffectors and that hyper-mechanical stimulation could up regulate focal contacts size as compared to hypo-mechanical that down regulate clusterization.

  16. Purine and pyrimidine nucleosides preserve human astrocytoma cell adenylate energy charge under ischemic conditions.

    PubMed

    Balestri, Francesco; Giannecchini, Michela; Sgarrella, Francesco; Carta, Maria Caterina; Tozzi, Maria Grazia; Camici, Marcella

    2007-02-01

    The brain depends on both glycolysis and mitochondrial oxidative phosphorylation for maintenance of ATP pools. Astrocytes play an integral role in brain functions providing trophic supports and energy substrates for neurons. In this paper, we report that human astrocytoma cells (ADF) undergoing ischemic conditions may use both purine and pyrimidine nucleosides as energy source to slow down cellular damage. The cells are subjected to metabolic stress conditions by exclusion of glucose and incubation with oligomycin (an inhibitor of oxidative phosphorylation). This treatment brings about a depletion of the ATP pool, with a concomitant increase in the AMP levels, which results in a significant decrease of the adenylate energy charge. The presence of purine nucleosides in the culture medium preserves the adenylate energy charge, and improves cell viability. Besides purine nucleosides, also pyrimidine nucleosides, such as uridine and, to a lesser extent, cytidine, are able to preserve the ATP pool. The determination of lactate in the incubation medium indicates that nucleosides can preserve the ATP pool through anaerobic glycolysis, thus pointing to a relevant role of the phosphorolytic cleavage of the N-glycosidic bond of nucleosides which generates, without energy expense, the phosphorylated pentose, which through the pentose phosphate pathway and glycolysis can be converted to energetic intermediates also in the absence of oxygen. In fact, ADF cells possess both purine nucleoside phosphorylase and uridine phosphorylase activities.

  17. Final Report - Durable Catalysts for Fuel Cell Protection during Transient Conditions

    SciTech Connect

    Atanasoski, Radoslav; van der Vliet, Dennis; Cullen, David; Atanasoska, Ljiljana

    2015-01-26

    The objective of this project was to develop catalysts that will enable proton exchange membranes (PEM) fuel cell systems to weather the damaging conditions in the fuel cell at voltages beyond the thermodynamic stability of water during the transient periods of start-up/shut-down and fuel starvation. Such catalysts are required to make it possible for the fuel cell to satisfy the 2015 DOE targets for performance and durability. The project addressed a key issue of importance for successful transition of PEM fuel cell technology from development to pre-commercial phase. This issue is the failure of the catalyst and the other thermodynamically unstable membrane electrode assembly (MEA) components during start-up/shut-down and local fuel starvation at the anode, commonly referred to as transient conditions. During these periods the electrodes can reach potentials higher than the usual 1.23V upper limit during normal operation. The most logical way to minimize the damage from such transient events is to minimize the potential seen by the electrodes. At lower positive potentials, increased stability of the catalysts themselves and reduced degradation of the other MEA components is expected.

  18. [SOS response of DNA repair and genetic cell instability under hypoxic conditions].

    PubMed

    Vasil'eva, S V; Strel'tsova, D A

    2011-01-01

    The SOS DNA repair pathway is induced in E. coli as a multifunctional cell response to a wide variety of signals: UV, X or gamma-irradiation, mitomycin C or nalidixic acid treatment, thymine starvation, etc. Triggering of the system can be used as a general and early sign of DNA damage. Additionally, the SOS-response is known to be an "error-prone" DNA repair pathway and one of the sources of genetic instability. Hypoxic conditions are established to be the major factor of genetic instability as well. In this paper we for the first time studied the SOS DNA repair response under hypoxic conditions induced by the well known aerobic SOS-inducers. The SOS DNA repair response was examined as a reaction of E. coli PQ37 [sfiA::lacZ] cells to UVC, NO-donating agents and 4NQO. Here we provide evidence that those agents were able to induce the SOS DNA repair response in E. coli at anaerobic growth conditions. The process does not depend on the transcriptional activity of the universal protein of E. col anaerobic growth Fnr [4Fe-4S]2+ or can not be referred to as an indicator of genetic instability in hypoxic conditions.

  19. Nanoparticle growth and surface chemistry changes in cell-conditioned culture medium

    PubMed Central

    Kendall, Michaela; Hodges, Nikolas J.; Whitwell, Harry; Tyrrell, Jess; Cangul, Hakan

    2015-01-01

    When biomolecules attach to engineered nanoparticle (ENP) surfaces, they confer the particles with a new biological identity. Physical format may also radically alter, changing ENP stability and agglomeration state within seconds. In order to measure which biomolecules are associated with early ENP growth, we studied ENPs in conditioned medium from A549 cell culture, using dynamic light scattering (DLS) and linear trap quadrupole electron transfer dissociation mass spectrometry. Two types of 100 nm polystyrene particles (one uncoated and one with an amine functionalized surface) were used to measure the influence of surface type. In identically prepared conditioned medium, agglomeration was visible in all samples after 1 h, but was variable, indicating inter-sample variability in secretion rates and extracellular medium conditions. In samples conditioned for 1 h or more, ENP agglomeration rates varied significantly. Agglomerate size measured by DLS was well correlated with surface sequestered peptide number for uncoated but not for amine coated polystyrene ENPs. Amine-coated ENPs grew much faster and into larger agglomerates associated with fewer sequestered peptides, but including significant sequestered lactose dehydrogenase. We conclude that interference with extracellular peptide balance and oxidoreductase activity via sequestration is worthy of further study, as increased oxidative stress via this new mechanism may be important for cell toxicity. PMID:25533102

  20. Bioluminescence Imaging of β Cells and Intrahepatic Insulin Gene Activity under Normal and Pathological Conditions

    PubMed Central

    Sekiguchi, Yukari; Nagasaki, Haruka; Daassi, Dhouha; Tai, Pei-Han; Ema, Masatsugu; Kudo, Takashi; Takahashi, Satoru

    2013-01-01

    In diabetes research, bioluminescence imaging (BLI) has been applied in studies of β-cell impairment, development, and islet transplantation. To develop a mouse model that enables noninvasive imaging of β cells, we generated a bacterial artificial chromosome (BAC) transgenic mouse in which a mouse 200-kbp genomic fragment comprising the insulin I gene drives luciferase expression (Ins1-luc BAC transgenic mouse). BLI of mice was performed using the IVIS Spectrum system after intraperitoneal injection of luciferin, and the bioluminescence signal from the pancreatic region analyzed. When compared with MIP-Luc-VU mice [FVB/N-Tg(Ins1-luc)VUPwrs/J] expressing luciferase under the control of the 9.2-kbp mouse insulin I promoter (MIP), the bioluminescence emission from Ins1-luc BAC transgenic mice was enhanced approximately 4-fold. Streptozotocin-treated Ins1-luc BAC transgenic mice developed severe diabetes concomitant with a sharp decline in the BLI signal intensity in the pancreas. Conversely, mice fed a high-fat diet for 8 weeks showed an increase in the signal, reflecting a decrease or increase in the β-cell mass. Although the bioluminescence intensity of the islets correlated well with the number of isolated islets in vitro, the intensity obtained from a living mouse in vivo did not necessarily reflect an absolute quantification of the β-cell mass under pathological conditions. On the other hand, adenovirus-mediated gene transduction of β-cell-related transcription factors in Ins1-luc BAC transgenic mice generated luminescence from the hepatic region for more than 1 week. These results demonstrate that BLI in Ins1-luc BAC transgenic mice provides a noninvasive method of imaging islet β cells and extrapancreatic activity of the insulin gene in the liver under normal and pathological conditions. PMID:23593212

  1. Lipophilic rather than hydrophilic photosensitizers show strong adherence to standard cell culture microplates under cell-free conditions.

    PubMed

    Engelhardt, Victoria; Kiesslich, Tobias; Berlanda, Juergen; Hofbauer, Stefanie; Krammer, Barbara; Plaetzer, Kristjan

    2011-06-02

    Analysis of photosensitizer (PS) uptake kinetics into tumor cells is a standard cell culture experiment in photodynamic therapy (PDT) - usually performed in plastic microplates or petri dishes. Organic substances such as PS can potentially interact with the plastic surfaces. In this study, we provide a qualitative comparison of three lipophilic PS (hypericin, Foscan® and Photofrin®) and two rather hydrophilic PS formulations (PVP-hypericin and aluminum (III) phthalocyanine tetrasulfonate chloride) regarding their adherence to the surfaces of 96-well microplates obtained from four different manufacturers. For estimation of the relevance of PS adherence for cellular uptake studies we compared the fluorescence signal of the respective PS in microplates containing A431 human epithelial carcinoma cells with microplates incubated with the respective PS under cell-free conditions. We demonstrate that lipophilic PS substances show a strong adherence to microplates - in case of direct lysis and fluorescence measurement resulting in 50% up to 90% of the overall signal to be caused by adherence of the substances to the plastic materials in a cellular uptake experiment. For the hydrophilic compounds, adherence is negligible. Interestingly, adherence of PS agents to microplates takes place in a time-dependent and thus kinetic-like manner, requiring up to several hours to reach a plateau of the fluorescence signal. Furthermore, PS adherence is a function of the PS concentration applied and no saturation effect was observed for the concentrations used in this study. Taken together, this study provides a systematic analysis under which conditions PS adherence to cell culture plates may contribute to the overall fluorescence signal in - for example - PS uptake experiments.

  2. Analysis of Proliferation of Melanoma Cells and Mesenchymal Stem Cells in Co-Culture and Contribution of Experimental Conditions into Interpretation of the Results.

    PubMed

    Kandarakov, O F; Kopantseva, E E; Belyavsky, A V

    2016-11-01

    A series of experiments on co-culturing of Mel IL melanoma cells and mesenhymal stem cells showed that these cells do not influence proliferation of each other, but we observed weaker adhesion of stromal stem cells to plastic in cocultures where with melanoma cells were grown on mesenhymal stem cells feeder. Cell proliferation was also considerably influenced by experimental conditions, which should be taken into account for correct interpretation of obtained results. The principles of experiments on co-culturing of cancer and stromal cells are formulated that take into account the most important factors influencing cell behavior and minimize the probability of artifact results. It was concluded that co-culturing conditions cells significantly affect the experimental results and can be the source of conflicting conclusions on mutual influence of stromal and cancer cells in vitro.

  3. Treosulfan-Based Conditioning Regimen in Sibling and Alternative Donor Hematopoietic Stem Cell Transplantation for Children with Sickle Cell Disease

    PubMed Central

    Marzollo, Antonio; Calore, Elisabetta; Tumino, Manuela; Pillon, Marta; Gazzola, Maria Vittoria; Destro, Roberta; Colombatti, Raffaella; Marson, Piero; Tison, Tiziana; Colpo, Anna; Mainardi, Chiara; Gabelli, Maria; Boaro, Maria Paola; Rossin, Sara; Strano, Aurora; Quaglia, Nadia; Menzato, Federica; Basso, Giuseppe; Sainati, Laura; Messina, Chiara

    2017-01-01

    Background and objectives Lack of suitable donors and regimen related toxicity are major barriers for hematopoietic stem cell transplantation (HSCT) in patients with sickle cell disease (SCD). The aim of the study is the assessment of efficacy and toxicity of Treosulfan-based conditioning regimen for SCD also when alternative donors such as mismatched unrelated donor and haploidentical donor are employed. Methods We report our single-center experience: 11 patients with SCD received HSCT with a Treosulfan/Thiotepa/Fludarabine/Anti-thymoglobulin conditioning regimen between 2010 and 2015. The donor was a matched sibling donor (n= 7), a haploidentical parent (n= 2), a matched unrelated donor (n= 1) or a mismatched unrelated donor (n=1). The haploidentical and mismatched unrelated donor grafts were manipulated by removing TCRαβ and CD19 positive cells. Results All patients survived the procedure and achieved stable engraftment. Stable mixed chimerism was observed in 5/11 patients. Grade III–IV regimen related toxicity was limited to mucositis and no grade III–IV graft-versus-host disease (GvHD) occurred. No SCD manifestation was observed post transplant and cerebral vasculopathy improved in 3/5 evaluable patients. Organ function evaluation showed no pulmonary, cardiac or renal toxicity but gonadal failure occurred in 1/4 evaluable patients. Conclusion Our data suggest that Treosulfan is associated with low toxicity and may be employed also for unrelated and haploidentical donor HSCT. PMID:28293402

  4. Condition medium of HepG-2 cells induces the transdifferentiation of human umbilical cord mesenchymal stem cells into cancerous mesenchymal stem cells

    PubMed Central

    Yang, Juan; Miao, Yinglei; Chang, Yefei; Zhang, Fan; Wang, Yubo; Zheng, Sheng

    2016-01-01

    This study aimed to investigate the transdifferentiation of human umbilical cord mesenchymal stem cells (hUCMSCs) into cancer-associated mesenchymal stem cells (CA-MSCs) after incubation with condition medium (CM) from liver cancer HepG-2 cells, and the biobehaviors (proliferation and migration) of these CA-MSCs were further evaluated. The supernatant of HepG-2 cells was collected and mixed with equal volume of low glucose DMEM. The resultant medium was used to treat hUCMSCs for 48 h. The expression of CA-MSCs related proteins and miR-221 was detected in cells. The supernatant of induced hUCMSCs was mixed with equal volume of high glucose DMEM, and the resultant medium was used treat HepG-2 cells for 48 h and the proliferation and migration of HepG-2 cells were evaluated. Moreover, HepG-2 cells were co-cultured with hUCMSCs and then the proliferation and migration of HepG-2 cells were assessed. After incubation with the supernatant from HepG-2 cells, hUCMSCs showed significantly elevated expression of vimentin, fibroblast activation protein (FAP) and miR-221. The supernatant of induced hUCMSCs was able to significantly increase the proliferation and migration of HepG-2 cells. Following co-culture, the proliferation and migration of HepG-2 cells increased dramatically. These findings suggest that the supernatant of HepG-2 cells is able to induce the phenotype of CA-MSCs and the supernatant of CA-MSCs may promote the proliferation and migration of HepG-2 cells. These findings provide experimental evidence for the cellular remodeling in tumor microenvironment and the safety of clinical use of hUCMSCs. PMID:27648133

  5. Decitabine has a biphasic effect on natural killer cell viability, phenotype, and function under proliferative conditions.

    PubMed

    Kopp, Lisa M; Ray, Anish; Denman, Cecele J; Senyukov, Vladimir S; Somanchi, Srinivas S; Zhu, Shiguo; Lee, Dean A

    2013-07-01

    DNA hypermethylation resulting in aberrant epigenetic silencing plays an important role in the oncogenesis of many cancer types, including acute myelogenous leukemia (AML).(4) The modulation of NK cell receptors and their cognate ligands is a known mechanism of immune escape in AML, and some membrane proteins, such as killer immunoglobulin-like receptors (KIR), are known to be transcriptionally regulated by DNA methylation of their promoter regions. Thus, restoring proper expression of immunoreceptors or their ligands with immunosensitizing drugs is an attractive approach to improving cancer immunotherapy. The cytidine analog 5-aza-2'-deoxycytidine (decitabine, DAC) has both a hypomethylating effect at low doses when incorporated into DNA and a cytotoxic effect at higher doses as a result of interfering with translation when incorporated into RNA. Thus, decitabine has been used at higher doses for its direct anti-leukemic effect, and is being tested at low doses for its ability to correct the malignant gene expression phenotype. A known benefit of hypomethylating agents is their ability to sensitize AML blasts to lysis by NK cells. However, there is little information on the direct effect of hypomethylating agents on NK cell phenotype, proliferation, survival, or function. We recently described a method for inducing robust proliferation of NK cells, enabling us to study the hypomethylating effects of decitabine. To distinguish direct toxicity of the decitabine from its hypomethylating effect, and promote hypomethylation during proliferation, decitabine was added to human peripheral blood NK cells at concentrations from 0.02 to 5μM under either static or proliferation-inducing culture conditions. After 5 days, NK cells were assessed for viability, proliferation, cytotoxicity, expression of major activating and inhibitory receptors, and global DNA methylation. Increasing concentrations of decitabine not only causes increased expression of KIR and the activating

  6. Receptor channel TRPC6 orchestrate the activation of human hepatic stellate cell under hypoxia condition

    SciTech Connect

    Iyer, Soumya C; Kannan, Anbarasu; Gopal, Ashidha; Devaraj, Niranjali; Halagowder, Devaraj

    2015-08-01

    Hepatic stellate cells (HSCs), a specialized stromal cytotype have a great impact on the biological behaviors of liver diseases. Despite this fact, the underlying mechanism that regulates HSC still remains poorly understood. The aim of the present study was to understand the role of TRPC6 signaling in regulating the molecular mechanism of HSCs in response to hypoxia. In the present study we showed that under hypoxia condition, the upregulated Hypoxia Inducible Factor 1α (HIF1α) increases NICD activation, which in turn induces the expression of transient receptor potential channel 6 (TRPC6) in HSC line lx-2. TRPC6 causes a sustained elevation of intracellular calcium which is coupled with the activation of the calcineurin-nuclear factor of activated T-cell (NFAT) pathway which activates the synthesis of extracellular matrix proteins. TRPC6 also activates SMAD2/3 dependent TGF-β signaling in facilitating upregulated expression of αSMA and collagen. As activated HSCs may be a suitable target for HCC therapy and targeting these cells rather than the HCC cells may result in a greater response. Collectively, our studies indicate for the first time the detailed mechanism of activation of HSC through TRPC6 signaling and thus being a promising therapeutic target. - Highlights: • HIF1α increases NICD, induces TRPC6 in lx2 cells. • TRPC6 a novel regulator in the activation of HSC. • HSCs as target for HCC therapy.

  7. Evaluation of the antineoplastic activity of gallic acid in oral squamous cell carcinoma under hypoxic conditions.

    PubMed

    Guimaraes, Talita A; Farias, Lucyana C; Fraga, Carlos A; Feltenberger, John D; Melo, Geraldo A; Coletta, Ricardo D; Souza Santos, Sergio H; de Paula, Alfredo M B; Guimaraes, Andre L

    2016-06-01

    The purpose of the current study was to develop and test a theoretical model that could explain the mechanism of action of gallic acid (GA) in the oral squamous cell carcinoma context for the first time. The theoretical model was developed using bioinformatics and interaction network analysis to evaluate the effect of GA on oral squamous cell carcinoma. In a second step to confirm theoretical results, migration, invasion, proliferation, and gene expression (Col1A1, E-cadherin, HIF-1α, and caspase-3) were performed under normoxic and hypoxic conditions. Our study indicated that treatment with GA resulted in the inhibition of cell proliferation, migration, and invasion in neoplastic cells. Observation of the molecular mechanism showed that GA upregulates E-cadherin expression and downregulates Col1A1 and HIF-1α expression, suggesting that GA might be a potential anticancer compound. In conclusion, the present study demonstrated that GA significantly reduces cell proliferation, invasion, and migration by increasing E-cadherin and repressing Col1A1.

  8. Conditioned media from differentiating craniofacial bone marrow stromal cells influence mineralization and proliferation in periodontal ligament stem cells.

    PubMed

    Jin, Zhenyu; Feng, Yuan; Liu, Hongwei

    2016-10-01

    Previous reports have mainly focused on the behavioral responses of human periodontal ligament stem cells (hPDLSCs) in interaction with tibia bone marrow stromal cells (BMSCs). However, there is little study on the biologic features of hPDLSCs under the induction of maxilla BMSCs (M-BMSCs) at different phases of osteogenic differentiation. We hypothesized that M-BMSCs undergoing osteogenic differentiation acted on the proliferation, differentiation, and bone-forming capacity of hPDLSCs. In this paper, primary hPDLSCs and human M-BMSCs (hM-BMSCs) were expanded in vitro. After screening of surface markers for characterization, hPDLSCs were cocultured with different phases of differentiating hM-BMSCs. Cell proliferation and alkaline phosphatase activity were examined, and mineralization-associated markers such as osteocalcin and runt-related transcription factor 2 of hPDLSCs in coculture with uninduced/osteoinduced hM-BMSCs were evaluated. hPDLSCs in hM-BMSCs-conditioned medium (hM-BMSCs-CM) group showed a reduction in proliferation compared with untreated hPDLSCs, while osteoinduced hM-BMSCs for 10 day-conditioned medium (hM-BMSCs-CM-10ds) and osteoinduced hM-BMSCs for 15 day-conditioned medium (hM-BMSCs-CM-15ds) enhance the proliferation of hPDLSCs. hM-BMSCs of separate differentiation stages temporarily inhibited osteogenesis of hPDLSCs in the early days. Upon extending time periods, uninduced/osteoinduced hM-BMSCs markedly enhanced osteogenesis of hPDLSCs to different degrees. The transplantation results showed hM-BMSCs-CM-15ds treatment promoted tissue regeneration to generate cementum/periodontal ligament-like structure characterized by hard-tissue formation. This research supported the notion that hM-BMSCs triggered osteogenesis of hPDLSCs suggesting important implications for periodontal engineering.

  9. Conception on the cell mechanisms of bone tissue loss under spase flight conditions

    NASA Astrophysics Data System (ADS)

    Rodionova, Natalia; Oganov, Victor; Kabitskaya, Olga

    Basing on the analysis of available literature and the results of our own electron microscopic and radioautographic researches the data are presented about the morpho-functional peculiarities and succession of cellular interactions in adaptive remodeling of bone structures under normal conditions and after exposure of animals (rats, monkeys, mice) to microgravity (SLS-2, Bion-11, BionM-1). The probable cellular mechanisms of the development of osteopenia and osteoporosis are considered. Our conception on remodeling proposes the following sequence in the development of cellular interactions after decrease of the mechanical loading: a primary response of osteocytes (mechanosensory cells) to the mechanical stimulus; osteocytic remodeling (osteolysis); transmission of the mechanical signals through a system of canals and processes to functionally active osteoblasts and surface osteocytes as well as to the bone-marrow stromal cells and to those lying on bone surfaces. As a response to the mechanical stimulus (microgravity) the system of stromal cell-preosteoblast-osteoblast shows a delay in proliferation, differentiation and specific functioning of the osteogenetic cells, some of the osteoblasts undergo apoptosis. Then the osteoclastic reaction occurs (attraction of monocytes and formation of osteoclasts and bone matrix resorption in the loci of apoptosis of osteoblasts and osteocytes). The macrophagal reaction is followed by osteoblastogenesis, which appears to be a rehabilitating process. However, during prolonged absence of mechanical stimuli (microgravity, long-time immobilization) the adaptive activization of osteoblastogenesis doesn’t occur (as it is the case during the physiological remodeling of bone tissue) or it occurs to a smaller degree. The loading deficit leads to an adaptive differentiation of stromal cells to fibroblastic cells and adipocytes in these remodeling loci. These cell reactions are considered as adaptive-compensatory, but they don’t result

  10. Metabolic analysis of antibody producing Chinese hamster ovary cell culture under different stresses conditions.

    PubMed

    Badsha, Md Bahadur; Kurata, Hiroyuki; Onitsuka, Masayoshi; Oga, Takushi; Omasa, Takeshi

    2016-07-01

    Chinese hamster ovary (CHO) cells are commonly used as the host cell lines concerning their ability to produce therapeutic proteins with complex post-translational modifications. In this study, we have investigated the time course extra- and intracellular metabolome data of the CHO-K1 cell line, under a control and stress conditions. The addition of NaCl and trehalose greatly suppressed cell growth, where the maximum viable cell density of NaCl and trehalose cultures were 2.2-fold and 2.8-fold less than that of a control culture. Contrariwise, the antibody production of both the NaCl and trehalose cultures was sustained for a longer time to surpass that of the control culture. The NaCl and trehalose cultures showed relatively similar dynamics of cell growth, antibody production, and substrate/product concentrations, while they indicated different dynamics from the control culture. The principal component analysis of extra- and intracellular metabolome dynamics indicated that their dynamic behaviors were consistent with biological functions. The qualitative pattern matching classification and hierarchical clustering analyses for the intracellular metabolome identified the metabolite clusters whose dynamic behaviors depend on NaCl and trehalose. The volcano plot revealed several reporter metabolites whose dynamics greatly change between in the NaCl and trehalose cultures. The elastic net identified some critical, intracellular metabolites that are distinct between the NaCl and trehalose. While a relatively small number of intracellular metabolites related to the cell growth, glucose, glutamine, lactate and ammonium ion concentrations, the mechanism of antibody production was suggested to be very complicated or not to be explained by elastic net regression analysis.

  11. Mesenchymal stem cell-conditioned medium prevents radiation-induced liver injury by inhibiting inflammation and protecting sinusoidal endothelial cells.

    PubMed

    Chen, Yi-Xing; Zeng, Zhao-Chong; Sun, Jing; Zeng, Hai-Ying; Huang, Yan-; Zhang, Zhen-Yu

    2015-07-01

    Current management of radiation-induced liver injury is limited. Sinusoidal endothelial cell (SEC) apoptosis and inflammation are considered to be initiating events in hepatic damage. We hypothesized that mesenchymal stem cells (MSCs) possess anti-apoptotic and anti-inflammatory actions during hepatic irradiation, acting via paracrine mechanisms. This study aims to examine whether MSC-derived bioactive components are protective against radiation-induced liver injury in rats. MSC-conditioned medium (MSC-CM) was generated from rat bone marrow-derived MSCs. The effect of MSC-CM on the viability of irradiated SECs was examined by flow cytometric analysis. Activation of the Akt and ERK pathways was analyzed by western blot. MSC-CM was also delivered to Sprague-Dawley rats immediately before receiving liver irradiation, followed by testing for pathological features, changes in serum hyaluronic acid, ALT, and inflammatory cytokine levels, and liver cell apoptosis. MSC-CM enhanced the viability of irradiated SECs in vitro and induced Akt and ERK phosphorylation in these cells. Infusion of MSC-CM immediately before liver irradiation provided a significant anti-apoptotic effect on SECs and improved the histopathological features of injury in the irradiated liver. MSC-CM also reduced the secretion and expression of inflammatory cytokines and increased the expression of anti-inflammatory cytokines. MSC-derived bioactive components could be a novel therapeutic approach for treating radiation-induced liver injury.

  12. Cellular interactions via conditioned media induce in vivo nephron generation from tubular epithelial cells or mesenchymal stem cells.

    PubMed

    Machiguchi, Toshihiko; Nakamura, Tatsuo

    2013-06-07

    There are some successful reports of kidney generation by utilizing the natural course of kidney development, namely, the use of an artificially treated metanephros, blastocyst or ureteric bud. Under a novel concept of cellular interactions via conditioned media (CMs), we have attempted in vivo nephron generation from tubular epithelial cells (TECs) or mesenchymal stem cells (MSCs). Here we used 10× CMs of vascular endothelial cells (VECs) and TECs, which is the first to introduce a CM into the field of organ regeneration. We first present stimulative cross-talks induced by these CMs between VECs and TECs on cell proliferation and morphological changes. In MSCs, TEC-CM suppressed these changes, however, induced cytokeratin expression, indicating the differentiation of MSCs into TECs. As a result, glomerular and tubular structures were created following the implantation of TECs or MSCs with both CMs. Our findings suggest that the cellular interactions via CMs might induce in vivo nephron generation from TECs or MSCs. As a promoting factor, CMs could also be applied to the regeneration of other organs and tissues.

  13. Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications

    PubMed Central

    Trombi, Luisa; Danti, Serena; Savelli, Sara; Moscato, Stefania; D'Alessandro, Delfo; Ricci, Claudio; Giannotti, Stefano; Petrini, Mario

    2016-01-01

    Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on potential biohazard and inflammation risks exerted by xenogeneic nutrients for their culture. Human derivatives or recombinant materials are the first choice candidates to reduce these reactions. Therefore, culture supplements and materials of autologous origin represent the best nutrients and the safest products. Here, we describe a new protocol for the isolation and culture of bone marrow hMSCs in autologous conditions — namely, patient-derived serum as a supplement for the culture medium and fibrin as a scaffold for hMSC administration. Indeed, hMSC/fibrin clot constructs could be extremely useful for several clinical applications. In particular, we focus on their use in orthopedic surgery, where the fibrin clot derived from the donor's own blood allowed effective cell delivery and nutrient/waste exchanges. To ensure optimal safety conditions, it is of the utmost importance to avoid the risks of hMSC transformation and tissue overgrowth. For these reasons, the approach described in this paper also indicates a minimally ex vivo hMSC expansion, to reduce cell senescence and morphologic changes, and short-term osteo-differentiation before implantation, to induce osteogenic lineage specification, thus decreasing the risk of subsequent uncontrolled proliferation. PMID:28060333

  14. Effects of Operating Conditions on Internal Resistances in Enzyme Fuel Cells Studied via Electrochemical Impedance Spectroscopy

    SciTech Connect

    Aaron, D; Borole, Abhijeet P; Yiacoumi, Sotira; Tsouris, Costas

    2012-01-01

    Enzyme fuel cells (EFCs) offer some advantages over traditional precious-metal-catalyzed fuel cells, such as polymer electrolyte membrane fuel cells (PEMFCs). However, EFCs exhibit far less power output than PEMFCs and have relatively short life spans before materials must be replaced. In this work, electrochemical impedance spectroscopy (EIS) is used to analyze the internal resistances throughout the EFC at a variety of operating conditions. EIS analysis is focused primarily on the resistances of the anode, solution/membrane, and cathode. Increased enzyme loading results in improved power output and reductions in internal resistance. Conditions are identified for which enzyme loading does not limit the EFC performance. EIS experiments are also reported for EFCs operated continuously for 2 days; power output declines sharply over time, while all internal resistances increase. Drying of the cathode and enzyme/mediator degradation are believed to have contributed to this behavior. Finally, experiments are performed at varying air-humidification temperatures. Little effect on internal resistances or power output is observed. However, it is anticipated that increased air humidification can improve longevity by delivering more water to the cathode. Improvements to the enzymatic cathode are needed for EFC development. These improvements need to focus on improving transport rather than increasing enzyme loading.

  15. Reduced-intensity conditioning allogeneic hematopoietic-cell transplantation for older patients with acute myeloid leukemia

    PubMed Central

    Goyal, Gaurav; Gundabolu, Krishna; Vallabhajosyula, Saraschandra; Silberstein, Peter T.; Bhatt, Vijaya Raj

    2016-01-01

    Elderly patients (>60 years) with acute myeloid leukemia have a poor prognosis with a chemotherapy-alone approach. Allogeneic hematopoietic-cell transplantation (HCT) can improve overall survival (OS). However, myeloablative regimens can have unacceptably high transplant-related mortality (TRM) in an unselected group of older patients. Reduced-intensity conditioning (RIC) or nonmyeloablative (NMA) conditioning regimens preserve the graft-versus-leukemia effects but reduce TRM. NMA regimens result in minimal cytopenia and may not require stem cell support for restoring hematopoiesis. RIC regimens, intermediate in intensity between NMA and myeloablative regimens, can cause prolonged myelosuppresion and usually require stem cell support. A few retrospective and prospective studies suggest a possibility of lower risk of relapse with myeloablative HCT in fit older patients with lower HCT comorbidity index; however, RIC and NMA HCTs have an important role in less-fit patients and those with significant comorbidities because of lower TRM. Whether early tapering of immunosuppression, monitoring of minimal residual disease, and post-transplant maintenance therapy can improve the outcomes of RIC and NMA HCT in elderly patients will require prospective trials. PMID:27247754

  16. Conditional Mutation of Rb Causes Cell Cycle Defects without Apoptosis in the Central Nervous System

    PubMed Central

    MacPherson, D.; Sage, J.; Crowley, D.; Trumpp, A.; Bronson, R. T.; Jacks, T.

    2003-01-01

    Targeted disruption of the retinoblastoma gene in mice leads to embryonic lethality in midgestation accompanied by defective erythropoiesis. Rb−/− embryos also exhibit inappropriate cell cycle activity and apoptosis in the central nervous system (CNS), peripheral nervous system (PNS), and ocular lens. Loss of p53 can prevent the apoptosis in the CNS and lens; however, the specific signals leading to p53 activation have not been determined. Here we test the hypothesis that hypoxia caused by defective erythropoiesis in Rb-null embryos contributes to p53-dependent apoptosis. We show evidence of hypoxia in CNS tissue from Rb−/− embryos. The Cre-loxP system was then used to generate embryos in which Rb was deleted in the CNS, PNS and lens, in the presence of normal erythropoiesis. In contrast to the massive CNS apoptosis in Rb-null embryos at embryonic day 13.5 (E13.5), conditional mutants did not have elevated apoptosis in this tissue. There was still significant apoptosis in the PNS and lens, however. Rb−/− cells in the CNS, PNS, and lens underwent inappropriate S-phase entry in the conditional mutants at E13.5. By E18.5, conditional mutants had increased brain size and weight as well as defects in skeletal muscle development. These data support a model in which hypoxia is a necessary cofactor in the death of CNS neurons in the developing Rb mutant embryo. PMID:12529408

  17. Phenotype overlap in glial cell populations: astroglia, oligodendroglia and NG-2(+) cells

    PubMed Central

    Alghamdi, Badrah; Fern, Robert

    2015-01-01

    The extent to which NG-2(+) cells form a distinct population separate from astrocytes is central to understanding whether this important cell class is wholly an oligodendrocyte precursor cell (OPC) or has additional functions akin to those classically ascribed to astrocytes. Early immuno-staining studies indicate that NG-2(+) cells do not express the astrocyte marker GFAP, but orthogonal reconstructions of double-labeled confocal image stacks here reveal a significant degree of co-expression in individual cells within post-natal day 10 (P10) and adult rat optic nerve (RON) and rat cortex. Extensive scanning of various antibody/fixation/embedding approaches identified a protocol for selective post-embedded immuno-gold labeling. This first ultrastructural characterization of identified NG-2(+) cells revealed populations of both OPCs and astrocytes in P10 RON. NG-2(+) astrocytes had classic features including the presence of glial filaments but low levels of glial filament expression were also found in OPCs and myelinating oligodendrocytes. P0 RONs contained few OPCs but positively identified astrocytes were observed to ensheath pre-myelinated axons in a fashion previously described as a definitive marker of the oligodendrocyte lineage. Astrocyte ensheathment was also apparent in P10 RONs, was absent from developing nodes of Ranvier and was never associated with compact myelin. Astrocyte processes were also shown to encapsulate some oligodendrocyte somata. The data indicate that common criteria for delineating astrocytes and oligodendroglia are insufficiently robust and that astrocyte features ascribed to OPCs may arise from misidentification. PMID:26106302

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

    PubMed

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

    2017-02-01

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

  19. Conditioned Medium from Adipose-Derived Stem Cells (ADSCs) Promotes Epithelial-to-Mesenchymal-Like Transition (EMT-Like) in Glioma Cells In vitro.

    PubMed

    Iser, Isabele C; Ceschini, Stefanie M; Onzi, Giovana R; Bertoni, Ana Paula S; Lenz, Guido; Wink, Márcia R

    2016-12-01

    Mesenchymal stem cells (MSCs) have recently been described to home to brain tumors and to integrate into the tumor-associated stroma. Understanding the communication between cancer cells and MSCs has become fundamental to determine whether MSC-tumor interactions should be exploited as a vehicle for therapeutic agents or considered a target for intervention. Therefore, we investigated whether conditioned medium from adipose-derived stem cells (ADSCs-CM) modulate glioma tumor cells by analyzing several cell biology processes in vitro. C6 rat glioma cells were treated with ADSCs-CM, and cell proliferation, cell cycle, cell viability, cell morphology, adhesion, migration, and expression of epithelial-mesenchymal transition (EMT)-related surface markers were analyzed. ADSCs-CM did not alter cell viability, cell cycle, and growth rate of C6 glioma cells but increased their migratory capacity. Moreover, C6 cells treated with ADSC-CM showed reduced adhesion and underwent changes in cell morphology. Up-regulation of EMT-associated markers (vimentin, MMP2, and NRAS) was also observed following treatment with ADSC-CM. Our findings demonstrate that the paracrine factors released by ADSCs are able to modulate glioma cell biology. Therefore, ADSC-tumor cell interactions in a tumor microenvironment must be considered in the design of clinical application of stem cell therapy. Graphical Abstract Factors released by adipose-derived stem cells (ADSCs) may modulate the biology of C6 glioma cells. When C6 cells are exposed to a conditioned medium from adipose-derived stem cells (ADSCs-CM), some of these cells can undergo an EMT-like process and trans-differentiate into cells with a more mesenchymal phenotype, characterized by enhanced expression of EMT-related surface markers, reduced cell adhesion capacity, increased migratory capacity, as well as changes in cell and nuclei morphology.

  20. Increased susceptibility to amyloid-β toxicity in rat brain microvascular endothelial cells under hyperglycemic conditions.

    PubMed

    Carvalho, Cristina; Katz, Paige S; Dutta, Somhrita; Katakam, Prasad V G; Moreira, Paula I; Busija, David W

    2014-01-01

    We hypothesized that hyperglycemia-induced mitochondrial dysfunction and oxidative stress are closely associated with amyloid-β peptide (Aβ) toxicity in endothelial cells. Brain microvascular endothelial cells from rat (RBMEC) and mice (MBMEC) were isolated from adult Sprague-Dawley rats and homozygous db/db (Leprdb/Leprdb) and heterozygous (Dock7m/Leprdb) mice, and cultured under normo- and hyperglycemic conditions for 7 d followed by 24 h exposure to Aβ1-40. Some experiments were also performed with two mitochondrial superoxide (O2•-) scavengers, MitoTempo and Peg-SOD. Cell viability was measured by the Alamar blue assay and mitochondrial membrane potential (ΔΨm) by confocal microscopy. Mitochondrial O2•- and hydrogen peroxide (H2O2) production was assessed by fluorescence microscopy and H2O2 production was confirmed by microplate reader. Hyperglycemia or Aβ1-40 alone did not affect cell viability in RBMEC. However, the simultaneous presence of high glucose and Aβ1-40 reduced cell viability and ΔΨm, and enhanced mitochondrial O2•- and H2O2 production. MitoTempo and PEG-SOD prevented Aβ1-40 toxicity. Interestingly, MBMEC presented a similar pattern of alterations with db/db cultures presenting higher susceptibility to Aβ1-40. Overall, our results show that high glucose levels increase the susceptibility of brain microvascular endothelial cells to Aβ toxicity supporting the idea that hyperglycemia is a major risk factor for vascular injury associated with AD.

  1. Conditioned medium from human amniotic epithelial cells may induce the differentiation of human umbilical cord blood mesenchymal stem cells into dopaminergic neuron-like cells.

    PubMed

    Yang, Shu; Sun, Hai-Mei; Yan, Ji-Hong; Xue, Hong; Wu, Bo; Dong, Fang; Li, Wen-Shuai; Ji, Feng-Qing; Zhou, De-Shan

    2013-07-01

    Dopaminergic (DA) neuron therapy has been established as a new clinical tool for treating Parkinson's disease (PD). Prior to cell transplantation, there are two primary issues that must be resolved: one is the appropriate seed cell origin, and the other is the efficient inducing technique. In the present study, human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) were used as the available seed cells, and conditioned medium from human amniotic epithelial cells (ACM) was used as the inducing reagent. Results showed that the proportion of DA neuron-like cells from hUCB-MSCs was significantly increased after cultured in ACM, suggested by the upregulation of DAT, TH, Nurr1, and Pitx3. To identify the process by which ACM induces DA neuron differentiation, we pretreated hUCB-MSCs with k252a, the Trk receptor inhibitor of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), and found that the proportion of DA neuron-like cells was significantly decreased compared with ACM-treated hUCB-MSCs, suggesting that NGF and BDNF in ACM were involved in the differentiation process. However, we could not rule out the involvement of other unidentified factors in the ACM, because ACM + k252a treatment does not fully block DA neuron-like cell differentiation compared with control. The transplantation of ACM-induced hUCB-MSCs could ameliorate behavioral deficits in PD rats, which may be associated with the survival of engrafted DA neuron-like cells. In conclusion, we propose that hUCB-MSCs are a good source of DA neuron-like cells and that ACM is a potential inducer to obtain DA neuron-like cells from hUCB-MSCs in vitro for an ethical and legal cell therapy for PD.

  2. Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning.

    PubMed

    Aiuti, Alessandro; Slavin, Shimon; Aker, Memet; Ficara, Francesca; Deola, Sara; Mortellaro, Alessandra; Morecki, Shoshana; Andolfi, Grazia; Tabucchi, Antonella; Carlucci, Filippo; Marinello, Enrico; Cattaneo, Federica; Vai, Sergio; Servida, Paolo; Miniero, Roberto; Roncarolo, Maria Grazia; Bordignon, Claudio

    2002-06-28

    Hematopoietic stem cell (HSC) gene therapy for adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID) has shown limited clinical efficacy because of the small proportion of engrafted genetically corrected HSCs. We describe an improved protocol for gene transfer into HSCs associated with nonmyeloablative conditioning. This protocol was used in two patients for whom enzyme replacement therapy was not available, which allowed the effect of gene therapy alone to be evaluated. Sustained engraftment of engineered HSCs with differentiation into multiple lineages resulted in increased lymphocyte counts, improved immune functions (including antigen-specific responses), and lower toxic metabolites. Both patients are currently at home and clinically well, with normal growth and development. These results indicate the safety and efficacy of HSC gene therapy combined with nonmyeloablative conditioning for the treatment of SCID.

  3. Molten Carbonate Fuel Cell performance analysis varying cathode operating conditions for carbon capture applications

    NASA Astrophysics Data System (ADS)

    Audasso, Emilio; Barelli, Linda; Bidini, Gianni; Bosio, Barbara; Discepoli, Gabriele

    2017-04-01

    The results of a systematic experimental campaign to verify the impact of real operating conditions on the performance of a complete Molten Carbonate Fuel Cell (MCFC) are presented. In particular, the effects of ageing and composition of water, oxygen and carbon dioxide in the cathodic feeding stream are studied through the analysis of current-voltage curves and Electrochemical Impedance Spectroscopy (EIS). Based on a proposed equivalent electrical circuit model and a fitting procedure, a correlation is found among specific operating parameters and single EIS coefficients. The obtained results suggest a new performance monitoring approach to be applied to MCFC for diagnostic purpose. Particular attention is devoted to operating conditions characteristic of MCFC application as CO2 concentrators, which, by feeding the cathode with exhaust gases, is a promising route for efficient and cheap carbon capture.

  4. Identifying the microbial communities and operational conditions for optimized wastewater treatment in microbial fuel cells.

    PubMed

    Ishii, Shun'ichi; Suzuki, Shino; Norden-Krichmar, Trina M; Wu, Angela; Yamanaka, Yuko; Nealson, Kenneth H; Bretschger, Orianna

    2013-12-01

    Microbial fuel cells (MFCs) are devices that exploit microorganisms as "biocatalysts" to recover energy from organic matter in the form of electricity. MFCs have been explored as possible energy neutral wastewater treatment systems; however, fundamental knowledge is still required about how MFC-associated microbial communities are affected by different operational conditions and can be optimized for accelerated wastewater treatment rates. In this study, we explored how electricity-generating microbial biofilms were established at MFC anodes and responded to three different operational conditions during wastewater treatment: 1) MFC operation using a 750 Ω external resistor (0.3 mA current production); 2) set-potential (SP) operation with the anode electrode potentiostatically controlled to +100 mV vs SHE (4.0 mA current production); and 3) open circuit (OC) operation (zero current generation). For all reactors, primary clarifier effluent collected from a municipal wastewater plant was used as the sole carbon and microbial source. Batch operation demonstrated nearly complete organic matter consumption after a residence time of 8-12 days for the MFC condition, 4-6 days for the SP condition, and 15-20 days for the OC condition. These results indicate that higher current generation accelerates organic matter degradation during MFC wastewater treatment. The microbial community analysis was conducted for the three reactors using 16S rRNA gene sequencing. Although the inoculated wastewater was dominated by members of Epsilonproteobacteria, Gammaproteobacteria, and Bacteroidetes species, the electricity-generating biofilms in MFC and SP reactors were dominated by Deltaproteobacteria and Bacteroidetes. Within Deltaproteobacteria, phylotypes classified to family Desulfobulbaceae and Geobacteraceae increased significantly under the SP condition with higher current generation; however those phylotypes were not found in the OC reactor. These analyses suggest that species

  5. Cell sourcing and culture conditions for fibrin-based valve constructs.

    PubMed

    Williams, Chrysanthi; Johnson, Sandra L; Robinson, Paul S; Tranquillo, Robert T

    2006-06-01

    Cell sourcing for tissue-engineered heart valves remains a critical issue. In this work, human dermal fibroblasts (HDF) or porcine valve interstitial cells (PVIC) were entrapped in adherent fibrin disk constructs and harvested at 3 and 5 weeks. We compared the fibrin remodeling abilities of each cell type in Dulbecco's Modified Eagle's Medium (DMEM) and DMEM/F12 supplemented with transforming growth factor beta (TGF), and the response of PVIC to DMEM/F12 supplemented with fibroblast growth factor (FGF), a combination of FGF and TGF, and TGF with varying ascorbic acid (AA) concentrations. Culture media were supplemented with serum, insulin, AA, a fibrinolysis inhibitor, and antibiotics. DMEM maximized collagen and elastin deposition by HDF, while DMEM/F12 with FGF yielded the highest fibrin remodeling response by PVIC. HDF degraded fibrin slower than PVIC, and PVIC constructs had higher cellularity than HDF constructs in DMEM and DMEM/F12 at 3 weeks. FGF addition increased collagen content, collagen deposited per cell, and collagen as percentage of total protein compared to medium supplemented with TGF or TGF and FGF. AA addition increased collagen deposition by PVIC, but there was no dose dependence between 50 and 150 microg/mL AA. These results collectively show that PVIC are able to remodel fibrin faster and exhibit greater mechanical stiffening compared to HDF. Conditions for increased collagen deposition are also identified toward the engineering of valve constructs.

  6. Real-time thermal imaging of solid oxide fuel cell cathode activity in working condition.

    PubMed

    Montanini, Roberto; Quattrocchi, Antonino; Piccolo, Sebastiano A; Amato, Alessandra; Trocino, Stefano; Zignani, Sabrina C; Faro, Massimiliano Lo; Squadrito, Gaetano

    2016-09-01

    Electrochemical methods such as voltammetry and electrochemical impedance spectroscopy are effective for quantifying solid oxide fuel cell (SOFC) operational performance, but not for identifying and monitoring the chemical processes that occur on the electrodes' surface, which are thought to be strictly related to the SOFCs' efficiency. Because of their high operating temperature, mechanical failure or cathode delamination is a common shortcoming of SOFCs that severely affects their reliability. Infrared thermography may provide a powerful tool for probing in situ SOFC electrode processes and the materials' structural integrity, but, due to the typical design of pellet-type cells, a complete optical access to the electrode surface is usually prevented. In this paper, a specially designed SOFC is introduced, which allows temperature distribution to be measured over all the cathode area while still preserving the electrochemical performance of the device. Infrared images recorded under different working conditions are then processed by means of a dedicated image processing algorithm for quantitative data analysis. Results reported in the paper highlight the effectiveness of infrared thermal imaging in detecting the onset of cell failure during normal operation and in monitoring cathode activity when the cell is fed with different types of fuels.

  7. Novel electrical energy storage system based on reversible solid oxide cells: System design and operating conditions

    NASA Astrophysics Data System (ADS)

    Wendel, C. H.; Kazempoor, P.; Braun, R. J.

    2015-02-01

    Electrical energy storage (EES) is an important component of the future electric grid. Given that no other widely available technology meets all the EES requirements, reversible (or regenerative) solid oxide cells (ReSOCs) working in both fuel cell (power producing) and electrolysis (fuel producing) modes are envisioned as a technology capable of providing highly efficient and cost-effective EES. However, there are still many challenges and questions from cell materials development to system level operation of ReSOCs that should be addressed before widespread application. This paper presents a novel system based on ReSOCs that employ a thermal management strategy of promoting exothermic methanation within the ReSOC cell-stack to provide thermal energy for the endothermic steam/CO2 electrolysis reactions during charging mode (fuel producing). This approach also serves to enhance the energy density of the stored gases. Modeling and parametric analysis of an energy storage concept is performed using a physically based ReSOC stack model coupled with thermodynamic system component models. Results indicate that roundtrip efficiencies greater than 70% can be achieved at intermediate stack temperature (680 °C) and elevated stack pressure (20 bar). The optimal operating condition arises from a tradeoff between stack efficiency and auxiliary power requirements from balance of plant hardware.

  8. Highly Efficient Neural Conversion of Human Pluripotent Stem Cells in Adherent and Animal-Free Conditions.

    PubMed

    Lukovic, Dunja; Diez Lloret, Andrea; Stojkovic, Petra; Rodríguez-Martínez, Daniel; Perez Arago, Maria Amparo; Rodriguez-Jimenez, Francisco Javier; González-Rodríguez, Patricia; López-Barneo, José; Sykova, Eva; Jendelova, Pavla; Kostic, Jelena; Moreno-Manzano, Victoria; Stojkovic, Miodrag; Bhattacharya, Shomi S; Erceg, Slaven

    2017-04-01

    Neural differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can produce a valuable and robust source of human neural cell subtypes, holding great promise for the study of neurogenesis and development, and for treating neurological diseases. However, current hESCs and hiPSCs neural differentiation protocols require either animal factors or embryoid body formation, which decreases efficiency and yield, and strongly limits medical applications. Here we develop a simple, animal-free protocol for neural conversion of both hESCs and hiPSCs in adherent culture conditions. A simple medium formula including insulin induces the direct conversion of >98% of hESCs and hiPSCs into expandable, transplantable, and functional neural progenitors with neural rosette characteristics. Further differentiation of neural progenitors into dopaminergic and spinal motoneurons as well as astrocytes and oligodendrocytes indicates that these neural progenitors retain responsiveness to instructive cues revealing the robust applicability of the protocol in the treatment of different neurodegenerative diseases. The fact that this protocol includes animal-free medium and human extracellular matrix components avoiding embryoid bodies makes this protocol suitable for the use in clinic. Stem Cells Translational Medicine 2017;6:1217-1226.

  9. Adaptation of CHO cells in serum-free conditions for erythropoietin production: Application of EVOP technique for process optimization.

    PubMed

    Jukić, Suzana; Bubenik, Dijana; Pavlović, Nediljko; Tušek, Ana Jurinjak; Srček, Višnja Gaurina

    2016-09-01

    Mammalian cell cultures are the preferred expression systems for the production of biopharmaceuticals requiring posttranslational processing. Usually, cell cultures are cultivated in medium supplemented with serum, which supports cell proliferation, viability, and productivity. However, due to scientific and regulatory concerns, serum-free conditions are required in recombinant protein production. Cell lines that are intended for commercial recombinant protein production have to adapt to serum- or protein-free conditions early in their development. This is a labor- and time-consuming process because of the specific cell requirements related to their adaptation in new microenvironment. In the present study, a Chinese hamster ovary (CHO) cell line producing glycosylated recombinant human erythropoietin (rhEPO) was adapted for growth and rhEPO production in serum- and protein-free conditions. The physiology, growth parameters, and morphology of the CHO cells and rhEPO biosynthesis and structure were closely monitored during the adaptation process to avoid unwanted selection of cell subpopulations. The results showed that the CHO cells were successfully adapted to suspension growth and rhEPO production in the protein-free conditions and that the structure of rhEPO remained nearly unchanged. In addition, during rhEPO production in the protein-free suspension conditions, the agitation rate seem to be significant for optimal process performance in contrast to the initial cell concentration, evaluated through evolutionary operation method.

  10. Maintenance of undifferentiated mouse embryonic stem cells in suspension by the serum- and feeder-free defined culture condition

    PubMed Central

    Tsuji, Yukiiko; Yoshimura, Naoko; Aoki, Hitomi; Sharov, Alexei A.; Ko, Minoru S.H.; Motohashi, Tsutomu; Kunisada, Takahiro

    2008-01-01

    The proven pluripotency of ES cells is expected to allow their therapeutic use for regenerative medicine. We present here a novel suspension culture method that facilitates the proliferation of pluripotent ES cells without feeder cells. The culture medium contains polyvinyl alcohol (PVA), free of either animal-derived or synthetic serum, and contains very low amounts of peptidic or proteinaceous materials, which are favorable for therapeutic use. ES cells showed sustained proliferation in the suspension culture, and their undifferentiated state and pluripotency were experimentally verified. DNA microarray analyses showed a close relationship between the elevated expression of genes related to cell adhesions. We suggest that this suspension culture condition provides a better alternative to the conventional attached cell culture condition, especially for possible therapeutic use, by limiting the exposure of ES cells to feeder cells and animal products. PMID:18624284

  11. Testicular cell-conditioned medium supports embryonic stem cell differentiation toward germ lineage and to spermatocyte- and oocyte-like cells.

    PubMed

    Shah, Syed M; Saini, Neha; Singh, Manoj K; Manik, Radheysham; Singla, Suresh K; Palta, Prabhat; Chauhan, Manmohan S

    2016-08-01

    Testicular cells are believed to secrete various growth factors that activate signaling pathways finally leading to gametogenesis. In vitro gametogenesis is an obscure but paramountly important task primarily because of paucity of the precursor cells and first trimester gonadal tissues. To overcome these limitations for development of in vitro gametes, the present study was designed to induce differentiation of buffalo embryonic stem (ES) cells into germ lineage cells on stimulation by testicular cell-conditioned medium (TCM), on the basis of the assumption that ES cells have the intrinsic property to differentiate into any cell type and TCM would provide the necessary growth factors for differentiation toward germ cell lineage. For this purpose, buffalo ES cells were differentiated as embryoid bodies (EB) in floating cultures and as monolayer adherent cultures in different doses (10%, 20%, and 40%) of TCM for different culture intervals (4, 8, and 14 days), to identify the optimum dose-and-time period. We observed that 40% TCM dose induces highest expression of primordial germ cell-specific (DAZL, VASA, and PLZF), meiotic (SYCP3, MLH1, TNP1/2, and PRM2), spermatocyte-specific (BOULE and TEKT1), and oocyte-specific genes (GDF9 and ZP2/3) for a culture period of 14 days under both floating and adherent differentiation. Immunocytochemical analysis of EBs and adherent cultures revealed presence of primordial germ cell markers (c-KIT, DAZL, and VASA), meiotic markers (SYCP3, MLH1 and PROTAMINE1), spermatocyte markers (ACROSIN and HAPRIN), and oocyte markers (GDF9 and ZP4), indicating progression into post-meiotic gametogenesis. The detection of germ cell-specific proteins in Day 14 EBs like VASA, GDF9, and ZP4 by Western blotting further confirmed germ lineage differentiation. The significantly lower (P < 0.05) concentration of 5-methyl-2-deoxycytidine in optimally differentiated EBs is suggestive of the process of methylation erasure. Oocyte-like structures

  12. Cell-cell and cell-surface interactions mediated by cellulose and a novel exopolysaccharide contribute to Pseudomonas putida biofilm formation and fitness under water-limiting conditions.

    PubMed

    Nielsen, Lindsey; Li, Xiaohong; Halverson, Larry J

    2011-05-01

    The composition of the exopolysaccharide matrix of Pseudomonas putida mt2 biofilms is relatively undefined as well as the contributions of each polymer to ecological fitness. Here, we describe the role of two putative exopolysaccharide gene clusters, putida exopolysaccharide A (pea) and bacterial cellulose (bcs) in biofilm formation and stability, rhizosphere colonization and matrix hydration under water-limiting conditions. Our findings suggest that pea is involved in the production of a novel glucose, galactose, and mannose-rich polymer that contributes to cell-cell interactions necessary for pellicle and biofilm formation and stability. In contrast, Bcs plays a minor role in biofilm formation and stability, although it does contribute to rhizosphere colonization based on a competition assay. We show that pea expression is highly induced transiently under water-limiting conditions but only slightly by high osmolarity, as determined by qRT-PCR. In contrast, both forms of water stress highly induced bcs expression. Cells deficient in making one or more exopolysaccharide experienced greater dehydration-mediated cell-envelope stress, leading to increased alginate promoter activity. However, this did not lead to increased exopolysaccharide production, except in bcs or pea mutants unable to produce alginate, indicating that P. putida compensates by producing, presumably more Pea or Bcs exopolysaccharides, to facilitate biofilm hydration. Collectively, the data suggest that Pea and Bcs contribute to biofilm formation and in turn their presence contributes to fitness under water-limiting conditions, but not to the extent of alginate.

  13. Lethal cutaneous disease in transgenic mice conditionally expressing type I human T cell leukemia virus Tax.

    PubMed

    Kwon, Hakju; Ogle, Louise; Benitez, Bobby; Bohuslav, Jan; Montano, Mauricio; Felsher, Dean W; Greene, Warner C

    2005-10-21

    Type I human T cell leukemia virus (HTLV-I) is etiologically linked with adult T cell leukemia, an aggressive and usually fatal expansion of activated CD4+ T lymphocytes that frequently traffic to skin. T cell transformation induced by HTLV-I involves the action of the 40-kDa viral Tax transactivator protein. Tax both stimulates the HTLV-I long terminal repeat and deregulates the expression of select cellular genes by altering the activity of specific host transcription factors, including cyclic AMP-responsive element-binding protein (CREB)/activating transcription factor, NF-kappaB/Rel, and serum response factor. To study initiating events involved in HTLV-I Tax-induced T cell transformation, we generated "Tet-off" transgenic mice conditionally expressing in a lymphocyte-restricted manner (EmuSR alpha promoter-enhancer) either wild-type Tax or mutant forms of Tax that selectively compromise the NF-kappaB (M22) or CREB/activating transcription factor (M47) activation pathways. Wild-type Tax and M47 Tax-expressing mice, but not M22-Tax expressing mice, developed progressive alopecia, hyperkeratosis, and skin lesions containing profuse activated CD4 T cell infiltrates with evidence of deregulated inflammatory cytokine production. In addition, these animals displayed systemic lymphadenopathy and splenomegaly. These findings suggest that Tax-mediated activation of NF-kappaB plays a key role in the development of this aggressive skin disease that shares several features in common with the skin disease occurring during the preleukemic stage in HTLV-I-infected patients. Of note, this skin disease completely resolved when Tax transgene expression was suppressed by administration of doxycycline, emphasizing the key role played by this viral oncoprotein in the observed pathology.

  14. Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

    PubMed Central

    Rao, Wei; Pan, Ning; Yang, Zhibo

    2016-01-01

    Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great potential for the detailed mass spectrometry (MS) analysis of biomolecules from biological samples. The single-probe, a miniaturized device with integrated sampling and ionization capabilities, is capable of performing both ambient MSI and in-situ SCMS analysis. For ambient MSI, the single-probe uses surface micro-extraction to continually conduct MS analysis of the sample, and this technique allows the creation of MS images with high spatial resolution (8.5 µm) from biological samples such as mouse brain and kidney sections. Ambient MSI has the advantage that little to no sample preparation is needed before the analysis, which reduces the amount of potential artifacts present in data acquisition and allows a more representative analysis of the sample to be acquired. For in-situ SCMS, the single-probe tip can be directly inserted into live eukaryotic cells such as HeLa cells, due to the small sampling tip size (< 10 µm), and this technique is capable of detecting a wide range of metabolites inside individual cells at near real-time. SCMS enables a greater sensitivity and accuracy of chemical information to be acquired at the single cell level, which could improve our understanding of biological processes at a more fundamental level than previously possible. The single-probe device can be potentially coupled with a variety of mass spectrometers for broad ranges of MSI and SCMS studies. PMID:27341402

  15. Differential responses of choroidal melanocytes and uveal melanoma cells to low oxygen conditions

    PubMed Central

    Weidmann, Cindy; Pomerleau, Jade; Trudel-Vandal, Laurence

    2017-01-01

    Purpose Tissue culture is traditionally performed at atmospheric oxygen concentration (21%), which induces hyperoxic stress, as endogenous physiologic oxygen tension found in tissues varies between 2% and 9%. This discrepancy may lead to misinterpretation of results and may explain why effects observed in vitro cannot always be reproduced in vivo and vice versa. Only a few studies have been conducted in low physiologic oxygen conditions to understand the development and differentiation of cells from the eye. Methods The aim of this study was to investigate the growth and gene expression profile of melanocytes from the choroid permanently exposed to 21% (hyperoxic) or 3% (physiologic) oxygen with proliferation assays and DNA microarray. The cellular behavior of the melanocytes was then compared to that of cancer cells. Results The gross morphology and melanin content of choroidal melanocytes changed slightly when they were exposed to 3% O2, and the doubling time was statistically significantly faster. There was an increase in the percentage of choroidal melanocytes in the active phases of the cell cycle as observed by using the proliferation marker Ki67. The caveolin-1 senescence marker was not increased in choroidal melanocytes or uveal melanoma cells grown in hyperoxia. In comparison, the morphology of the uveal melanoma cells was similar between the two oxygen levels, and the doubling time was slower at 3% O2. Surprisingly, gene expression profiling of the choroidal melanocytes did not reveal a large list of transcripts considerably dysregulated between the two oxygen concentrations; only the lactate transporter monocarboxylate transporter (MCT4) was statistically significantly upregulated at 3% O2. Conclusions This study showed that the oxygen concentration must be tightly controlled in experimental settings, because it influences the subsequent cellular behavior of human choroidal melanocytes. PMID:28356703

  16. Conditioned media cause increases in select osteogenic and adipogenic differentiation markers in mesenchymal stem cell cultures.

    PubMed

    Maxson, Scott; Burg, Karen J L

    2008-01-01

    The optimal mesenchymal stem cell (MSC) culture conditions that would allow clinically viable tissue-engineered devices are still yet to be determined. Most MSCs are found in the bone marrow, an area that also contains numerous osteoblasts and adipocytes. Paracrine signalling may be leveraged to modulate MSC differentiation in the preparation of a tissue-engineered device. Thus, the objectives of this study were to determine the effects of adipocyte-conditioned medium (CM) on MSC differentiation to osteoblasts and to determine the effects of osteoblast CM on MSC differentiation to adipocytes. Two groups of murine MSCs were given either an osteogenic differentiation medium or an adipogenic differentiation medium. CM was taken from one group and administered to the opposite group in concentrations of 25% or 50%. Metabolic activity, total protein and alkaline phosphatase (ALP) assays were conducted on the osteogenic group at predefined time points throughout the 21 day study, while metabolic activity, triglyceride and oil red O assays were conducted on the adipogenic group at predefined time points. Adipocyte CM administered at a concentration of 50% increased the ALP production of MSCs undergoing osteogenic differentiation. Additionally, osteoblast CM increased the triglyceride production of MSCs undergoing adipogenic differentiation and enlarged the lipid vesicles that were produced by the cells. The effects of the osteoblast CM were seen at both concentrations, but were greatest at the 50% CM level.

  17. Photoinduced Interfacial Electron Injection Dynamics in Dye-Sensitized Solar Cells under Photovoltaic Operating Conditions.

    PubMed

    Teuscher, Joël; Décoppet, Jean-David; Punzi, Angela; Zakeeruddin, Shaik M; Moser, Jacques-E; Grätzel, Michael

    2012-12-20

    We report a pump-probe spectroscopy study of electron injection rates in dye-sensitized solar cell (DSSC) devices. We examine the case of working devices employing an N719 ruthenium sensitizer and an iodide electrolyte. Electron injection is found to occur mainly on a sub-100 fs time scale, followed by a slower component with a lifetime of 26.9 ps, in accordance with previous reports on model samples. The amplitude of this latter component varies with electrolyte composition from 25 to 9%. The appearance of slower components in the electron injection dynamics may be attributed to an aggregated or weakly bound state of the surface-adsorbed N719 sensitizer. Further measurements are reported varying the cell light bias and load conditions, revealing no influence on electron injection dynamics. No other electron injection event is found to occur up to 1 ns. These results show no evidence for a slowdown of electron injection under working conditions compared to model systems for the electrolytes examined in this study.

  18. Effects of operating conditions on durability of polymer electrolyte membrane fuel cell Pt cathode catalyst layer

    NASA Astrophysics Data System (ADS)

    Ohyagi, Shinsuke; Matsuda, Toshihiko; Iseki, Yohei; Sasaki, Tatsuyoshi; Kaito, Chihiro

    In this study, we investigated the effects of humidity and oxygen reduction on the degradation of the catalyst of a polymer electrolyte membrane fuel cell (PEMFC) in a voltage cycling test. To elucidate the effect of humidity on the voltage cycling corrosion of a carbon-supported Pt catalyst with 3 nm Pt particles, voltage cycling tests based on 10,000 cycles were conducted using 100% relative humidity (RH) hydrogen as anode gas and nitrogen of varying humidities as cathode gas. The degradation rate of an electrochemical surface area (ECSA) was almost 50% under 189% RH nitrogen atmosphere and the Pt average particle diameter after 10,000 cycles under these conditions was about 2.3 times that of a particle of fresh catalyst because of the agglomeration of Pt particles. The oxygen reduction reaction (ORR) that facilitated Pt catalyst agglomeration when oxygen was employed as the cathode gas also demonstrated that Pt agglomeration was prominent in higher concentrations of oxygen. The ECSA degradation figure in 100% RH oxygen was similar to that in 189% RH nitrogen. It was concluded that liquid water, which was dropped under a supersaturated condition or generated by ORR, accelerated Pt agglomeration. In this paper, we suggest that the Pt agglomeration degradation occurs in a flooding area in a cell plane.

  19. Stabilization of Pt monolayer catalysts under harsh conditions of fuel cells

    SciTech Connect

    Zhang, Xiaoming; Yu, Shansheng; Zheng, Weitao E-mail: pingliu3@bnl.gov; Qiao, Liang; Liu, Ping E-mail: pingliu3@bnl.gov

    2015-05-21

    We employed density functional theory to explore the stability of core (M = Cu, Ru, Rh, Pd, Ag, Os, Ir, Au)-shell (Pt) catalysts under harsh conditions, including solutions and reaction intermediates involved in the oxygen reduction reaction (ORR) in fuel cells. A pseudomorphic surface alloy (PSA) with a Pt monolayer (Pt{sub 1ML}) supported on an M surface, Pt{sub 1ML}/M(111) or (001), was considered as a model system. Different sets of candidate M cores were identified to achieve a stable Pt{sub 1ML} shell depending on the conditions. In vacuum conditions, the Pt{sub 1ML} shell can be stabilized on the most of M cores except Cu, Ag, and Au. The situation varies under various electrochemical conditions. Depending on the solutions and the operating reaction pathways of the ORR, different M should be considered. Pd and Ir are the only core metals studied, being able to keep the Pt{sub ML} shell intact in perchloric acid, sulfuric acid, phosphoric acid, and alkaline solutions as well as under the ORR conditions via different pathways. Ru and Os cores should also be paid attention, which only fall during the ORR via the *OOH intermediate. Rh core works well as long as the ORR does not undergo the pathway via *O intermediate. Our results show that PSAs can behave differently from the near surface alloy, Pt{sub 1ML}/M{sub 1ML}/Pt(111), highlighting the importance of considering both chemical environments and the atomic structures in rational design of highly stable core-shell nanocatalysts. Finally, the roles that d-band center of a core M played in determining the stability of supported Pt{sub 1ML} shell were also discussed.

  20. Stabilization of Pt monolayer catalysts under harsh conditions of fuel cells

    SciTech Connect

    Zhang, Xiaoming; Liu, Ping; Yu, Shansheng; Qiao, Liang; Zheng, Weitao

    2015-05-21

    We employed density functional theory (DFT) to explore the stability of core (M = Cu, Ru, Rh, Pd, Ag, Os, Ir, Au)-shell (Pt) catalysts under harsh conditions, including solutions and reaction intermediates involved in the oxygen reduction reaction (ORR) in fuel cells. A pseudomorphic surface alloy (PSA) with a Pt monolayer (Pt1ML) supported on an M surface, Pt1ML/M(111) or (001), was considered as a model system. Different sets of candidate M cores were identified to achieve a stable Pt1ML shell depending on the conditions. In vacuum conditions, the Pt1ML shell can be stabilized on the most of M cores except Cu, Ag, and Au. The situation varies under various electrochemical conditions. Depending on the solutions and the operating reaction pathways of the ORR, different M should be considered. Pd and Ir are the only core metals studied, being able to keep the PtML shell intact in perchloric acid, sulfuric acid, phosphoric acid, and alkaline solutions as well as under the ORR conditions via different pathways. Ru and Os cores should also be paid attention, which only fall during the ORR via the *OOH intermediate. Rh core works well as long as the ORR does not undergo the pathway via *O intermediate. Our results show that PSAs can behave differently from the near surface alloy, Pt1ML/M1ML/Pt(111), highlighting the importance of considering both chemical environments and the atomic structures in rational design of highly stable core-shell nanocatalysts. Finally, the roles that d-band center of a core M played in determining the stability of supported Pt1ML shell were also discussed.

  1. Stabilization of Pt monolayer catalysts under harsh conditions of fuel cells

    NASA Astrophysics Data System (ADS)

    Zhang, Xiaoming; Yu, Shansheng; Qiao, Liang; Zheng, Weitao; Liu, Ping

    2015-05-01

    We employed density functional theory to explore the stability of core (M = Cu, Ru, Rh, Pd, Ag, Os, Ir, Au)-shell (Pt) catalysts under harsh conditions, including solutions and reaction intermediates involved in the oxygen reduction reaction (ORR) in fuel cells. A pseudomorphic surface alloy (PSA) with a Pt monolayer (Pt1ML) supported on an M surface, Pt1ML/M(111) or (001), was considered as a model system. Different sets of candidate M cores were identified to achieve a stable Pt1ML shell depending on the conditions. In vacuum conditions, the Pt1ML shell can be stabilized on the most of M cores except Cu, Ag, and Au. The situation varies under various electrochemical conditions. Depending on the solutions and the operating reaction pathways of the ORR, different M should be considered. Pd and Ir are the only core metals studied, being able to keep the PtML shell intact in perchloric acid, sulfuric acid, phosphoric acid, and alkaline solutions as well as under the ORR conditions via different pathways. Ru and Os cores should also be paid attention, which only fall during the ORR via the *OOH intermediate. Rh core works well as long as the ORR does not undergo the pathway via *O intermediate. Our results show that PSAs can behave differently from the near surface alloy, Pt1ML/M1ML/Pt(111), highlighting the importance of considering both chemical environments and the atomic structures in rational design of highly stable core-shell nanocatalysts. Finally, the roles that d-band center of a core M played in determining the stability of supported Pt1ML shell were also discussed.

  2. Stabilization of Pt monolayer catalysts under harsh conditions of fuel cells

    DOE PAGES

    Zhang, Xiaoming; Liu, Ping; Yu, Shansheng; ...

    2015-05-21

    We employed density functional theory (DFT) to explore the stability of core (M = Cu, Ru, Rh, Pd, Ag, Os, Ir, Au)-shell (Pt) catalysts under harsh conditions, including solutions and reaction intermediates involved in the oxygen reduction reaction (ORR) in fuel cells. A pseudomorphic surface alloy (PSA) with a Pt monolayer (Pt1ML) supported on an M surface, Pt1ML/M(111) or (001), was considered as a model system. Different sets of candidate M cores were identified to achieve a stable Pt1ML shell depending on the conditions. In vacuum conditions, the Pt1ML shell can be stabilized on the most of M cores exceptmore » Cu, Ag, and Au. The situation varies under various electrochemical conditions. Depending on the solutions and the operating reaction pathways of the ORR, different M should be considered. Pd and Ir are the only core metals studied, being able to keep the PtML shell intact in perchloric acid, sulfuric acid, phosphoric acid, and alkaline solutions as well as under the ORR conditions via different pathways. Ru and Os cores should also be paid attention, which only fall during the ORR via the *OOH intermediate. Rh core works well as long as the ORR does not undergo the pathway via *O intermediate. Our results show that PSAs can behave differently from the near surface alloy, Pt1ML/M1ML/Pt(111), highlighting the importance of considering both chemical environments and the atomic structures in rational design of highly stable core-shell nanocatalysts. Finally, the roles that d-band center of a core M played in determining the stability of supported Pt1ML shell were also discussed.« less

  3. The differentiation potential of gingival mesenchymal stem cells induced by apical tooth germ cell-conditioned medium

    PubMed Central

    Chen, Yan; Liu, Hongwei

    2016-01-01

    Gingival-derived mesenchymal stem cells (GMSCs) have recently been harvested; however, the use of GMSCs in periodontal tissue engineering requires further study. The present study established an indirect co-culture system between rat apical tooth germ-conditioned medium (APTG-CM) and GMSCs, in order to determine the effects on periodontal tissue differentiation in vitro and in vivo. Using the limiting dilution technique, single-colony derived human GMSCs and periodontal ligament stem cells (PDLSCs) were isolated and expanded to obtain homogeneous populations. PDLSCs were used as a positive control group. Cell cycle distribution, alkaline phosphatase (ALP) activity, mineralization behavior, expression of genes associated with a cementoblast phenotype (osteocalcin, bone sialoprotein, ALP, type I collagen, cementum-derived protein 23), and in vivo differentiation capacities of GMSCs/PDLSCs co-cultured with APTG-CM were evaluated. Flow cytometry indicated that GMSCs and PDLSCs were positive for STRO-1 and CD105, whereas CD45 expression was negative. The cell types were capable of forming colonies, and of osteogenic and adipogenic differentiation in response to appropriate stimuli. The induced GMSCs and PDLSCs exhibited numerous characteristics associated with cementoblast lineages, as indicated by increased proliferation and ALP activity, and upregulated expression of cementum-associated genes in vitro. In vivo, cementum/periodontal ligament-like structures were shown to form along the dentin surface and ceramic bovine bone in GMSCs and PDLSCs induced by APTG-CM group. Conversely, vertical fibers could not insert in the control group, which was not co-cultured with APTG-CM. In conclusion, GMSCs are likely to have a role in periodontal tissue regeneration. In addition, APTG-CM was able to provide a cementogenic microenvironment and promote differentiation of GMSCs along the cementoblastic lineage. PMID:27600358

  4. Development and Maturation of the Neuromuscular Junciton in Cell Culture Under Conditions of Simulated Zero-gravity

    NASA Technical Reports Server (NTRS)

    Gruener, R.

    1985-01-01

    Alterations in gravitational conditions which alter the normal development and interactions of nerve and muscle cells grown in culture is examined. Clinostat conditions, similating Og, which produce changes in cell morphology and growth patterns is studied. Data show that rotation of cocultures of nerve and muscle cells results in morphologic changes which are predicted to significantly alter the functional interactions between the elements of a prototypic synapse. It is further predicted that similar alterations may occur in central synapses which may therefore affect the development of the central nervous system when subjected to altered gravitational conditions.

  5. Cell-Autonomous Progeroid Changes in Conditional Mouse Models for Repair Endonuclease XPG Deficiency

    PubMed Central

    Vermeij, Wilbert P.; Tresini, Maria; Weymaere, Michael; Menoni, Hervé; Brandt, Renata M. C.; de Waard, Monique C.; Botter, Sander M.; Sarker, Altaf H.; Jaspers, Nicolaas G. J.; van der Horst, Gijsbertus T. J.; Cooper, Priscilla K.; Hoeijmakers, Jan H. J.; van der Pluijm, Ingrid

    2014-01-01

    As part of the Nucleotide Excision Repair (NER) process, the endonuclease XPG is involved in repair of helix-distorting DNA lesions, but the protein has also been implicated in several other DNA repair systems, complicating genotype-phenotype relationship in XPG patients. Defects in XPG can cause either the cancer-prone condition xeroderma pigmentosum (XP) alone, or XP combined with the severe neurodevelopmental disorder Cockayne Syndrome (CS), or the infantile lethal cerebro-oculo-facio-skeletal (COFS) syndrome, characterized by dramatic growth failure, progressive neurodevelopmental abnormalities and greatly reduced life expectancy. Here, we present a novel (conditional) Xpg−/− mouse model which -in a C57BL6/FVB F1 hybrid genetic background- displays many progeroid features, including cessation of growth, loss of subcutaneous fat, kyphosis, osteoporosis, retinal photoreceptor loss, liver aging, extensive neurodegeneration, and a short lifespan of 4–5 months. We show that deletion of XPG specifically in the liver reproduces the progeroid features in the liver, yet abolishes the effect on growth or lifespan. In addition, specific XPG deletion in neurons and glia of the forebrain creates a progressive neurodegenerative phenotype that shows many characteristics of human XPG deficiency. Our findings therefore exclude that both the liver as well as the neurological phenotype are a secondary consequence of derailment in other cell types, organs or tissues (e.g. vascular abnormalities) and support a cell-autonomous origin caused by the DNA repair defect itself. In addition they allow the dissection of the complex aging process in tissue- and cell-type-specific components. Moreover, our data highlight the critical importance of genetic background in mouse aging studies, establish the Xpg−/− mouse as a valid model for the severe form of human XPG patients and segmental accelerated aging, and strengthen the link between DNA damage and aging. PMID:25299392

  6. Studies on penetration of antibiotic in bacterial cells in space conditions (7-IML-1)

    NASA Technical Reports Server (NTRS)

    Tixador, R.

    1992-01-01

    The Cytos 2 experiment was performed aboard Salyut 7 in order to test the antibiotic sensitivity of bacteria cultivated in vitro in space. An increase of the Minimal Inhibitory Concentration (MIC) in the inflight cultures (i.e., an increase of the antibiotic resistance) was observed. Complementary studies of the ultrastructure showed a thickening of the cell envelope. In order to confirm the results of the Cytos 2 experiment, we performed the ANTIBIO experiment during the D1 mission to try to differentiate, by means of the 1 g centrifuge in the Biorack, between the biological effects of cosmic rays and those caused by microgravity conditions. The originality of this experiment was in the fact that it was designed to test the antibiotic sensitivity of bacteria cultivated in vitro during the orbital phase of the flight. The results show an increase in resistance to Colistin in in-flight bacteria. The MIC is practically double in the in-flight cultures. A cell count of living bacteria in the cultures containing the different Colistin concentrations showed a significant difference between the cultures developed during space flight and the ground based cultures. The comparison between the 1 g and 0 g in-flight cultures show similar behavior for the two sets. Nevertheless, a small difference between the two sets of ground based control cultures was noted. The cultures developed on the ground centrifuge (1.4 g) present a slight decrease in comparison with the cultures developed in the static rack (1 g). In order to approach the mechanisms of the increase of antibiotic resistance on bacteria cultivated in vitro in space, we have proposed the study on penetration of antibiotics in bacterial cells in space conditions. This experiment was selected for the International Microgravity Laboratory 1 (IML-1) mission.

  7. Cell-autonomous progeroid changes in conditional mouse models for repair endonuclease XPG deficiency

    SciTech Connect

    Barnhoorn, Sander; Uittenboogaard, Lieneke M.; Jaarsma, Dick; Vermeij, Wilbert P.; Tresini, Maria; Weymaere, Michael; Menoni, Hervé; Brandt, Renata M. C.; de Waard, Monique C.; Botter, Sander M.; Sarker, Altaf H.; Jaspers, Nicolaas G. J.; van der Horst, Gijsbertus T. J.; Cooper, Priscilla K.; Hoeijmakers, Jan H. J.; van der Pluijm, Ingrid; Niedernhofer, Laura J.

    2014-10-09

    As part of the Nucleotide Excision Repair (NER) process, the endonuclease XPG is involved in repair of helix-distorting DNA lesions, but the protein has also been implicated in several other DNA repair systems, complicating genotype-phenotype relationship in XPG patients. Defects in XPG can cause either the cancer-prone condition xeroderma pigmentosum (XP) alone, or XP combined with the severe neurodevelopmental disorder Cockayne Syndrome (CS), or the infantile lethal cerebro-oculo-facio-skeletal (COFS) syndrome, characterized by dramatic growth failure, progressive neurodevelopmental abnormalities and greatly reduced life expectancy. Here, we present a novel (conditional) Xpg-/- mouse model which—in a C57BL6/FVB F1 hybrid genetic background—displays many progeroid features, including cessation of growth, loss of subcutaneous fat, kyphosis, osteoporosis, retinal photoreceptor loss, liver aging, extensive neurodegeneration, and a short lifespan of 4–5 months. We show that deletion of XPG specifically in the liver reproduces the progeroid features in the liver, yet abolishes the effect on growth or lifespan. In addition, specific XPG deletion in neurons and glia of the forebrain creates a progressive neurodegenerative phenotype that shows many characteristics of human XPG deficiency. Our findings therefore exclude that both the liver as well as the neurological phenotype are a secondary consequence of derailment in other cell types, organs or tissues (e.g. vascular abnormalities) and support a cell-autonomous origin caused by the DNA repair defect itself. In addition they allow the dissection of the complex aging process in tissue- and cell-type-specific components. Moreover, our data highlight the critical importance of genetic background in mouse aging studies, establish the Xpg-/- mouse as a valid model for the severe form of human XPG patients and segmental accelerated aging, and strengthen the link between DNA damage and aging.

  8. Cell-autonomous progeroid changes in conditional mouse models for repair endonuclease XPG deficiency

    DOE PAGES

    Barnhoorn, Sander; Uittenboogaard, Lieneke M.; Jaarsma, Dick; ...

    2014-10-09

    As part of the Nucleotide Excision Repair (NER) process, the endonuclease XPG is involved in repair of helix-distorting DNA lesions, but the protein has also been implicated in several other DNA repair systems, complicating genotype-phenotype relationship in XPG patients. Defects in XPG can cause either the cancer-prone condition xeroderma pigmentosum (XP) alone, or XP combined with the severe neurodevelopmental disorder Cockayne Syndrome (CS), or the infantile lethal cerebro-oculo-facio-skeletal (COFS) syndrome, characterized by dramatic growth failure, progressive neurodevelopmental abnormalities and greatly reduced life expectancy. Here, we present a novel (conditional) Xpg-/- mouse model which—in a C57BL6/FVB F1 hybrid genetic background—displays manymore » progeroid features, including cessation of growth, loss of subcutaneous fat, kyphosis, osteoporosis, retinal photoreceptor loss, liver aging, extensive neurodegeneration, and a short lifespan of 4–5 months. We show that deletion of XPG specifically in the liver reproduces the progeroid features in the liver, yet abolishes the effect on growth or lifespan. In addition, specific XPG deletion in neurons and glia of the forebrain creates a progressive neurodegenerative phenotype that shows many characteristics of human XPG deficiency. Our findings therefore exclude that both the liver as well as the neurological phenotype are a secondary consequence of derailment in other cell types, organs or tissues (e.g. vascular abnormalities) and support a cell-autonomous origin caused by the DNA repair defect itself. In addition they allow the dissection of the complex aging process in tissue- and cell-type-specific components. Moreover, our data highlight the critical importance of genetic background in mouse aging studies, establish the Xpg-/- mouse as a valid model for the severe form of human XPG patients and segmental accelerated aging, and strengthen the link between DNA damage and aging.« less

  9. Cardiosphere conditioned media influence the plasticity of human mediastinal adipose tissue-derived mesenchymal stem cells.

    PubMed

    Siciliano, Camilla; Chimenti, Isotta; Ibrahim, Mohsen; Napoletano, Chiara; Mangino, Giorgio; Scafetta, Gaia; Zoccai, Giuseppe Biondi; Rendina, Erino Angelo; Calogero, Antonella; Frati, Giacomo; De Falco, Elena

    2015-01-01

    Nowadays, cardiac regenerative medicine is facing many limitations because of the complexity to find the most suitable stem cell source and to understand the regenerative mechanisms involved. Mesenchymal stem cells (MSCs) have shown great regenerative potential due to their intrinsic properties and ability to restore cardiac functionality, directly by transdifferentiation and indirectly by paracrine effects. Yet, how MSCs could respond to definite cardiac-committing microenvironments, such as that created by resident cardiac progenitor cells in the form of cardiospheres (CSs), has never been addressed. Recently, a putative MSC pool has been described in the mediastinal fat (hmADMSCs), but both its biology and function remain hitherto unexplored. Accordingly, we investigated the potential of hmADMSCs to be committed toward a cardiovascular lineage after preconditioning with CS-conditioned media (CCM). Results indicated that CCM affects cell proliferation. Gene expression levels of multiple cardiovascular and stemness markers (MHC, KDR, Nkx2.5, Thy-1, c-kit, SMA) are significantly modulated, and the percentage of hmADMSCs preconditioned with CCM and positive for Nkx2.5, MHC, and KDR is significantly higher relative to FBS and explant-derived cell conditioned media (EDCM, the unselected stage before CS formation). Growth factor-specific and survival signaling pathways (i.e., Erk1/2, Akt, p38, mTOR, p53) present in CCM are all equally regulated. Nonetheless, earlier BAD phosphorylation (Ser112) occurs associated with the CS microenvironment (and to a lesser extent to EDCM), whereas faster phosphorylation of PRAS40 in FBS, and of Akt (Ser473) in EDCM and 5-azacytidine occurs compared to CCM. For the first time, we demonstrated that the MSC pool held in the mediastinal fat is adequately plastic to partially differentiate in vitro toward a cardiac-like lineage. Besides, we have provided novel evidence of the potent inductive niche-like microenvironment that the CS

  10. Modeling and short circuit detection of 18650 Li-ion cells under mechanical abuse conditions

    NASA Astrophysics Data System (ADS)

    Sahraei, Elham; Campbell, John; Wierzbicki, Tomasz

    2012-12-01

    In this research a simple, yet accurate model of a single cell, needed for safety assessment of batteries under mechanical abuse conditions, was developed. Extensive testing was performed on a 18650 lithium ion cell, including indentation by a hemispherical punch, lateral indentation by a cylindrical rod, compression between two flat plates, and three-point bending. The batteries were tested in an environmental chamber at a 10% SOC. A finite element model was developed, composed of shell elements representing outside casing, and solid elements for the active material with a binder lumped together with the current collectors and the separator. The jelly roll is modeled as a homogenized and isotropic material. The homogenous model assumes different properties in tension and compression, but does not account for the effect of structural anisotropy caused by the layered nature of the jelly roll. Very good correlation was obtained between LS Dyna numerical simulation and test results in terms of load-displacement relations, deformed shape of the battery, and initiation and propagation of a crack in the shell casing. The FE model was also capable of predicting the onset of short circuit of the cell.

  11. Quantitative analysis of tryptase- and chymase-containing mast cells in eosinophilic conditions of cats.

    PubMed

    Noli, C; Welle, M; Scarampella, F; Abramo, F

    2003-03-01

    The presence and density of tryptase-positive/chymase-positive mast cells (MCs) (MC(TC)), chymase-positive/tryptase-negative MCs (MCC), and tryptase-positive/chymase-negative MCs (MC(T)) in lesional skin from cats with eosinophilic conditions were investigated. Skin biopsy specimens from eight cats with eosinophilic plaque (three cats), eosinophilic granuloma (two cats), and eosinophilic dermatitis (three cats) were studied. Toluidine blue staining and a double-enzyme-immunohistochemical staining technique were performed to determine MC density and MC subtypes, respectively. MC density varied from 170.3 to 503 cells/mm2 (mean value of 314.9 cells/mm2). In the superficial dermis, 5.9% of the MC belonged to the MC(T), 12.8% to the MC(C), and 81.2% to the MC(TC) subtype. In the deep dermis, 12.8% belonged to the MC(T), 12.8% to the MC(C), and 73.8% to the MC(TC) subtype. It is the first time that MC(C) have been identified. The double-labeling procedure proved to be a reliable tool for identifying simultaneously the presence of MC subtypes in feline skin.

  12. Beneficial effects of adiponectin on periodontal ligament cells under normal and regenerative conditions.

    PubMed

    Nokhbehsaim, Marjan; Keser, Sema; Nogueira, Andressa Vilas Boas; Cirelli, Joni Augusto; Jepsen, Søren; Jäger, Andreas; Eick, Sigrun; Deschner, James

    2014-01-01

    Type 2 diabetes and obesity are increasing worldwide and linked to periodontitis, a chronic disease which is characterized by the irreversible destruction of the tooth-supporting tissues, that is, periodontium. The mechanisms underlying the association of diabetes mellitus and obesity with periodontal destruction and compromised periodontal healing are not well understood, but decreased plasma levels of adiponectin, as found in diabetic and obese individuals, might be a critical mechanistic link. The aim of this in vitro study was to examine the effects of adiponectin on periodontal ligament (PDL) cells under normal and regenerative conditions, and to study the regulation of adiponectin and its receptors in these cells. Adiponectin stimulated significantly the expression of growth factors and extracellular matrix, proliferation, and in vitro wound healing, reduced significantly the constitutive tumor necrosis factor-α expression, and caused a significant upregulation of its own expression. The beneficial actions of enamel matrix derivative on a number of PDL cell functions critical for periodontal regeneration were partially enhanced by adiponectin. The periodontopathogen Porphyromonas gingivalis inhibited the adiponectin expression and stimulated the expression of its receptors. In conclusion, reduced levels of adiponectin, as found in type 2 diabetes and obesity, may compromise periodontal health and healing.

  13. Conditioned medium from mesenchymal stem cells induces cell death in organotypic cultures of rat hippocampus and aggravates lesion in a model of oxygen and glucose deprivation.

    PubMed

    Horn, Ana Paula; Frozza, Rudimar Luiz; Grudzinski, Patrícia Benke; Gerhardt, Daniéli; Hoppe, Juliana Bender; Bruno, Alessandra Nejar; Chagastelles, Pedro; Nardi, Nance Beyer; Lenz, Guido; Salbego, Christianne

    2009-01-01

    Cell therapy using bone marrow-derived mesenchymal stem cells (MSC) seems to be a new alternative for the treatment of neurological diseases, including stroke. In order to investigate the response of hippocampal tissue to factors secreted by MSC and if these factors are neuroprotective in a model of oxygen and glucose deprivation (OGD), we used organotypic hippocampal cultures exposed to conditioned medium from bone marrow-derived MSC. Our results suggest that the conditioned medium obtained from these cells aggravates lesion caused by OGD. In addition, the presence of the conditioned medium alone was toxic mainly to cells in the CA1, CA2 and CA3 areas of the hippocampal organotypic culture even in basal conditions. GABA stimulation and NMDA and AMPA receptors antagonists were able to reduce propidium iodide staining, suggesting that the cell death induced by the toxic factors secreted by MSC could involve these receptors.

  14. Secretion of nerve growth factor, brain-derived neurotrophic factor, and glial cell-line derived neurotrophic factor in co-culture of four cell types in cerebrospinal fluid-containing medium.

    PubMed

    Feng, Sanjiang; Zhuang, Minghua; Wu, Rui

    2012-12-25

    The present study co-cultured human embryonic olfactory ensheathing cells, human Schwann cells, human amniotic epithelial cells and human vascular endothelial cells in complete culture medium-containing cerebrospinal fluid. Enzyme linked immunosorbent assay was used to detect nerve growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor secretion in the supernatant of co-cultured cells. Results showed that the number of all cell types reached a peak at 7-10 days, and the expression of nerve growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor peaked at 9 days. Levels of secreted nerve growth factor were four-fold higher than brain-derived neurotrophic factor, which was three-fold higher than glial cell line-derived neurotrophic factor. Increasing concentrations of cerebrospinal fluid (10%, 20% and 30%) in the growth medium caused a decrease of neurotrophic factor secretion. Results indicated co-culture of human embryonic olfactory ensheathing cells, human Schwann cells, human amniotic epithelial cells and human vascular endothelial cells improved the expression of nerve growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor. The reduction of cerebrospinal fluid extravasation at the transplant site after spinal cord injury is beneficial for the survival and secretion of neurotrophic factors from transplanted cells.

  15. Long-term cultures of stem/progenitor cells from lobular and ductal breast carcinomas under non-adherent conditions

    PubMed Central

    Nardone, Agostina; Corvigno, Sara; Brescia, Annalisa; D’Andrea, Daniel; Limite, Gennaro

    2010-01-01

    A small subpopulation of stem/progenitor cells can give rise to the diversity of differentiated cells that comprise the bulk of the tumor. Are proliferating cells, within the bulk of tumor, few cells with uncommon features? The cell biological approach provides a limitless model for studying the hierarchical organization of progenitor subpopulation and identifying potential therapeutic targets. Aim of the study was to expand patients’ breast cancer cells for evaluating functional cell properties, and to characterize the protein expression profile of selected cells to be compared with that of primary tumors. Breast cancer cells from estrogen receptor (ERα) positive, HER2 negative lobular (LoBS cells) and ductal (DuBS cells) histotype were cultured under non-adherent conditions to form mammospheres. Sorting of the cells by their surface expression of CD24 and CD44 gave rise to subpopulations which were propagated, enriched and characterized for the expression of epithelial and stromal markers. We found that non-adherent culture conditions generate mammospheres of slowly proliferating cells; single cells, dissociated from mammospheres, grow in soft agar; long-term cultured LoBS and DuBS cells, CD44+/CD24low, express cytokeratin 5 (CK5), α-smooth muscle actin (α-sma) and vimentin, known as markers of basal/myoepithelial cells; and ERα (only DuBS cells), HER1 (EGF-Receptor), activated HER2, and cyclinD1 as markers of luminal epithelial cell. Isolates of cells from breast cancer patients may be a tool for a marker-driven testing of targeted therapies. PMID:21188518

  16. Compositional changes in 'Bartlett' pear ( Pyrus communis L.) cell wall polysaccharides as affected by sunlight conditions.

    PubMed

    Raffo, María D; Ponce, Nora M A; Sozzi, Gabriel O; Vicente, Ariel R; Stortz, Carlos A

    2011-11-23

    Preharvest conditions can have a great impact on fruit quality attributes and postharvest responses. Firmness is an important quality attribute in pear, and excessive softening increases susceptibility to bruising and decay, thus limiting fruit postharvest life. Textural characteristics of fruits are determined at least in part by cell wall structure and disassembly. Few studies have analyzed the influence of fruit preharvest environment in softening, cell wall composition, and degradation. In the current work 'Bartlett' pears grown either facing the sun (S) or in the shade (H) were harvested and stored for 13 days at 20 °C. An evaluation of fruit soluble solids, acidity, color, starch degradation, firmness, cell wall yield, pectin and matrix glycan solubilization, depolymerization, and monosaccharide composition was carried out. Sun-exposed pears showed more advanced color development and similar levels of starch degradation, sugars, and acids than shaded fruit. Sunlight-grown pears were at harvest firmer than shade-grown pears. Both fruit groups softened during storage at 20 °C, but even after ripening, sun-exposed pears remained firmer. Sunlight exposure did not have a great impact on pectin molecular weight. Instead, at harvest a higher proportion of water-solubilized uronic acids and alkali-solubilized neutral sugars and a larger mean molecular size of tightly bound glycans was found in sun-exposed pears. During ripening cell wall catabolism took place in both sun- and shade-grown pears, but pectin solubilization was clearly delayed in sun-exposed fruit. This was associated with decreased removal of RG I-arabinan side chains rather than with reduced depolymerization.

  17. Impact of conditioning intensity and TBI on acute GVHD after hematopoietic cell transplantation.

    PubMed

    Nakasone, H; Fukuda, T; Kanda, J; Mori, T; Yano, S; Kobayashi, T; Miyamura, K; Eto, T; Kanamori, H; Iwato, K; Uchida, N; Mori, S; Nagamura-Inoue, T; Ichinohe, T; Atsuta, Y; Teshima, T; Murata, M

    2015-04-01

    The impact of the conditioning intensity and TBI on acute GVHD (aGVHD) is still a matter of debate. We analyzed 6848 adult recipients who received allogeneic hematopoietic cell transplants (HCT) between 2006 and 2011 in Japan. The subjects were divided into groups who had received myeloablative conditioning (MAC) or reduced-intensity conditioning (RIC), either with or without TBI. There was a significant difference in the incidence of aGVHD 2-4 among the different conditioning types: 39% in TBI-MAC, 35% in TBI-RIC and 32% in both no-TBI MAC and no-TBI-RIC (P<0.001). In a multivariate analysis, TBI-MAC, but not no-TBI MAC, was significantly associated with an increased risk of aGVHD 2-4 (hazard ratio (HR) 1.33, P<0.01), whereas TBI-RIC was associated with an increased risk of GVHD 3-4 (HR 1.36, P=0.048). TBI-MAC and TBI-RIC were significantly associated with skin and gastrointestinal aGVHD. Subgroup analyses demonstrated that not only TBI-MAC, but also TBI-RIC, was significantly associated with aGVHD 2-4 in older patients. Furthermore, high-dose TBI only had an adverse impact on aGVHD 2-4 in HLA-matched HCT. Impacts of intensity and TBI on aGVHD differ by patient backgrounds, and this difference should be considered to establish a risk-adapted strategy for the prevention of aGVHD.

  18. Relating Cerebellar Purkinje Cell Activity to the Timing and Amplitude of Conditioned Eyelid Responses

    PubMed Central

    Khilkevich, Andrei; Mauk, Michael D.

    2015-01-01

    How Purkinje cell (PC) activity may be altered by learning is central to theories of the cerebellum. Pavlovian eyelid conditioning, because of how directly it engages the cerebellum, has helped reveal many aspects of cerebellar learning and the underlying mechanisms. Theories of cerebellar learning assert that climbing fiber inputs control plasticity at synapses onto PCs, and thus PCs control the expression of learned responses. We tested this assertion by recording 184 eyelid PCs and 240 non-eyelid PCs during the expression of conditioned eyelid responses (CRs) in well trained rabbits. By contrasting the responses of eyelid and non-eyelid PCs and by contrasting the responses of eyelid PCs under conditions that produce differently timed CRs, we test the hypothesis that learning-related changes in eyelid PCs contribute to the learning and adaptive timing of the CRs. We used a variety of analyses to test the quantitative relationships between eyelid PC responses and the kinematic properties of the eyelid CRs. We find that the timing of eyelid PC responses varies systematically with the timing of the behavioral CRs and that there are differences in the magnitude of eyelid PC responses between larger-CR, smaller-CR, and non-CR trials. However, eyelid PC activity does not encode any single kinematic property of the behavioral CRs at a fixed time lag, nor does it linearly encode CR amplitude. Even so, the results are consistent with the hypothesis that learning-dependent changes in PC activity contribute to the adaptively timed expression of conditioned eyelid responses. PMID:25995469

  19. Melatonin promotes development of haploid germ cells from early developing spermatogenic cells of Suffolk sheep under in vitro condition.

    PubMed

    Deng, Shou-Long; Chen, Su-Ren; Wang, Zhi-Peng; Zhang, Yan; Tang, Ji-Xin; Li, Jian; Wang, Xiu-Xia; Cheng, Jin-Mei; Jin, Cheng; Li, Xiao-Yu; Zhang, Bao-Lu; Yu, Kun; Lian, Zheng-Xing; Liu, Guo-Shi; Liu, Yi-Xun

    2016-05-01

    Promotion of spermatogonial stem cell (SSC) differentiation into functional sperms under in vitro conditions is a great challenge for reproductive physiologists. In this study, we observed that melatonin (10(-7) M) supplementation significantly enhanced the cultured SSCs differentiation into haploid germ cells. This was confirmed by the expression of sperm special protein, acrosin. The rate of SSCs differentiation into sperm with melatonin supplementation was 11.85 ± 0.93% which was twofold higher than that in the control. The level of testosterone, the transcriptions of luteinizing hormone receptor (LHR), and the steroidogenic acute regulatory protein (StAR) were upregulated with melatonin treatment. At the early stage of SSCs culture, melatonin suppressed the level of cAMP, while at the later stage, it promoted cAMP production. The similar pattern was observed in testosterone content. Expressions for marker genes of meiosis anaphase, Dnmt3a, and Bcl-2 were upregulated by melatonin. In contrast, Bax expression was downregulated. Importantly, the in vitro-generated sperms were functional and they were capable to fertilize oocytes. These fertilized oocytes have successfully developed to the blastula stage.

  20. Spontaneous follicular exclusion of SHP1-deficient B cells is conditional on the presence of competitor wild-type B cells.

    PubMed

    Schmidt, K N; Hsu, C W; Griffin, C T; Goodnow, C C; Cyster, J G

    1998-03-16

    Engagement of antigen receptors on mature B lymphocytes is known to block cell entry into lymphoid follicles and promote accumulation in T cell zones, yet the molecular basis for this change in cell distribution is not understood. Previous studies have shown that follicular exclusion requires a threshold level of antigen receptor engagement combined with occupancy of follicles by B cells without equivalent receptor engagement. The possibility has been raised that follicular composition affects B cell positioning by altering the amount of available antigen and the degree of receptor occupancy. Here we show that follicular composition affects migration of mature B cells under conditions that are independent of antigen receptor occupancy. B cells deficient in the negative regulatory protein tyrosine phosphatase, SHP1, which have elevated intracellular signaling by the B cell receptor, are shown to accumulate in the T zone in the absence of their specific antigen. Follicular exclusion of SHP1-deficient B cells was found to be conditional on the presence of excess B cells that lack elevated intracellular signaling, and was not due to a failure of SHP-1-deficient cells to mature and express the follicle-homing chemokine receptor Burkitt's lymphoma receptor 1. These findings strongly suggest that signals that are negatively regulated by SHP1 promote B cell localization in T cell zones by reducing competitiveness for follicular entry, and provide further evidence that follicular composition influences the positioning of antigen-engaged B cells.

  1. Trophic effects of mesenchymal stem cells in chondrocyte co-cultures are independent of culture conditions and cell sources.

    PubMed

    Wu, Ling; Prins, Henk-Jan; Helder, Marco N; van Blitterswijk, Clemens A; Karperien, Marcel

    2012-08-01

    Earlier, we have shown that the increased cartilage production in pellet co-cultures of chondrocytes and bone marrow-derived mesenchymal stem cells (BM-MSCs) is due to a trophic role of the MSC in stimulating chondrocyte proliferation and matrix production rather than MSCs actively undergoing chondrogenic differentiation. These studies were performed in a culture medium that was not compatible with the chondrogenic differentiation of MSCs. In this study, we tested whether the trophic role of the MSCs is dependent on culturing co-culture pellets in a medium that is compatible with the chondrogenic differentiation of MSCs. In addition, we investigated whether the trophic role of the MSCs is dependent on their origins or is a more general characteristic of MSCs. Human BM-MSCs and bovine primary chondrocytes were co-cultured in a medium that was compatible with the chondrogenic differentiation of MSCs. Enhanced matrix production was confirmed by glycosaminoglycans (GAG) quantification. A species-specific quantitative polymerase chain reaction demonstrated that the cartilage matrix was mainly of bovine origin, indicative of a lack of the chondrogenic differentiation of MSCs. In addition, pellet co-cultures were overgrown by bovine cells over time. To test the influence of origin on MSCs' trophic effects, the MSCs isolated from adipose tissue and the synovial membrane were co-cultured with human primary chondrocytes, and their activity was compared with BM-MSCs, which served as control. GAG quantification again confirmed increased cartilage matrix production, irrespective of the source of the MSCs. EdU staining combined with cell tracking revealed an increased proliferation of chondrocytes in each condition. Irrespective of the MSC source, a short tandem repeat analysis of genomic DNA showed a decrease in MSCs in the co-culture over time. Our results clearly demonstrate that in co-culture pellets, the MSCs stimulate cartilage formation due to a trophic effect on the

  2. Tumor Associated Stromal Cells Play a Critical Role on the Outcome of the Oncolytic Efficacy of Conditionally Replicative Adenoviruses

    PubMed Central

    Lopez, M. Verónica; Viale, Diego L.; Cafferata, Eduardo G. A.; Bravo, Alicia I.; Carbone, Cecilia; Gould, David; Chernajovsky, Yuti; Podhajcer, Osvaldo L.

    2009-01-01

    The clinical efficacy of conditionally replicative oncolytic adenoviruses (CRAd) is still limited by the inefficient infection of the tumor mass. Since tumor growth is essentially the result of a continuous cross-talk between malignant and tumor-associated stromal cells, targeting both cell compartments may profoundly influence viral efficacy. Therefore, we developed SPARC promoter-based CRAds since the SPARC gene is expressed both in malignant cells and in tumor-associated stromal cells. These CRAds, expressing or not the Herpes Simplex thymidine kinase gene (Ad-F512 and Ad(I)-F512-TK, respectively) exerted a lytic effect on a panel of human melanoma cells expressing SPARC; but they were completely attenuated in normal cells of different origins, including fresh melanocytes, regardless of whether cells expressed or not SPARC. Interestingly, both CRAds displayed cytotoxic activity on SPARC positive-transformed human microendothelial HMEC-1 cells and WI-38 fetal fibroblasts. Both CRAds were therapeutically effective on SPARC positive-human melanoma tumors growing in nude mice but exhibited restricted efficacy in the presence of co-administered HMEC-1 or WI-38 cells. Conversely, co-administration of HMEC-1 cells enhanced the oncolytic efficacy of Ad(I)-F512-TK on SPARC-negative MIA PaCa-2 pancreatic cancer cells in vivo. Moreover, conditioned media produced by stromal cells pre-infected with the CRAds enhanced the in vitro viral oncolytic activity on pancreatic cancer cells, but not on melanoma cells. The whole data indicate that stromal cells might play an important role on the outcome of the oncolytic efficacy of conditionally replicative adenoviruses. PMID:19337591

  3. Novel 3-D cell culture system for in vitro evaluation of anticancer drugs under anchorage-independent conditions.

    PubMed

    Aihara, Ayako; Abe, Natsuki; Saruhashi, Koichiro; Kanaki, Tatsuro; Nishino, Taito

    2016-12-01

    Anticancer drug discovery efforts have used 2-D cell-based assay models, which fail to forecast in vivo efficacy and result in a lower success rate of clinical approval. Recent 3-D cell culture models are expected to bridge the gap between 2-D and in vivo models. However, 3-D cell culture methods that are available for practical anticancer drug screening have not yet been fully attained. In this study, we screened several polymers for their ability to suspend cells or cell spheroids homogeneously in a liquid medium without changing the viscosity behavior, and identified gellan gum (FP001), as the most potent polymer. FP001 promoted cell dispersion in the medium and improved the proliferation of a wide range of cancer cell lines under low attachment conditions by inhibiting the formation of large-sized spheroids. In addition, cancer cells cultured with FP001-containing medium were more susceptible to inhibitors of epidermal growth factor (EGF) signaling than those cultured under attachment conditions. We also showed that ligands of the EGF receptor family clearly enhance proliferation of SKOV3 ovarian carcinoma cells under anchorage-independent conditions with FP001. Consistent with this result, the cells grown with FP001 showed higher EGF receptor content compared with cells cultured under attachment conditions. In conclusion, we developed a novel 3-D cell culture system that is available for high throughput screening of anticancer agents, and is suitable for evaluation of molecular-targeted anticancer drugs. Three-dimensional cell culture using FP001 will be of value in the development of useful technologies for anticancer drug discovery.

  4. Local area water removal analysis of a proton exchange membrane fuel cell under gas purge conditions.

    PubMed

    Lee, Chi-Yuan; Lee, Yu-Ming; Lee, Shuo-Jen

    2012-01-01

    In this study, local area water content distribution under various gas purging conditions are experimentally analyzed for the first time. The local high frequency resistance (HFR) is measured using novel micro sensors. The results reveal that the liquid water removal rate in a membrane electrode assembly (MEA) is non-uniform. In the under-the-channel area, the removal of liquid water is governed by both convective and diffusive flux of the through-plane drying. Thus, almost all of the liquid water is removed within 30 s of purging with gas. However, liquid water that is stored in the under-the-rib area is not easy to remove during 1 min of gas purging. Therefore, the re-hydration of the membrane by internal diffusive flux is faster than that in the under-the-channel area. Consequently, local fuel starvation and membrane degradation can degrade the performance of a fuel cell that is started from cold.

  5. N-cadherin is Key to Expression of the Nucleus Pulposus Cell Phenotype under Selective Substrate Culture Conditions

    PubMed Central

    Hwang, Priscilla Y; Jing, Liufang; Chen, Jun; Lim, Foon-Lian; Tang, Ruhang; Choi, Hyowon; Cheung, Kenneth M; Risbud, Makarand V; Gersbach, Charles A; Guilak, Farshid; Leung, Victor Y; Setton, Lori A

    2016-01-01

    Nucleus pulposus (NP) cells of the intervertebral disc are essential for synthesizing extracellular matrix that contributes to disc health and mechanical function. NP cells have a unique morphology and molecular expression pattern derived from their notochordal origin, and reside in N-cadherin (CDH2) positive cell clusters in vivo. With disc degeneration, NP cells undergo morphologic and phenotypic changes including loss of CDH2 expression and ability to form cell clusters. Here, we investigate the role of CDH2 positive cell clusters in preserving healthy, biosynthetically active NP cells. Using a laminin-functionalized hydrogel system designed to mimic features of the native NP microenvironment, we demonstrate NP cell phenotype and morphology is preserved only when NP cells form CDH2 positive cell clusters. Knockdown (CRISPRi) or blocking CDH2 expression in vitro and in vivo results in loss of a healthy NP cell. Findings also reveal that degenerate human NP cells that are CDH2 negative can be promoted to re-express CDH2 and healthy, juvenile NP matrix synthesis patterns by promoting cell clustering for controlled microenvironment conditions. This work also identifies CDH2 interactions with β-catenin-regulated signaling as one mechanism by which CDH2-mediated cell interactions can control NP cell phenotype and biosynthesis towards maintenance of healthy intervertebral disc tissues. PMID:27292569

  6. Differential sulfation remodelling of heparan sulfate by extracellular 6-O-sulfatases regulates fibroblast growth factor-induced boundary formation by glial cells: implications for glial cell transplantation.

    PubMed

    Higginson, Jennifer R; Thompson, Sophie M; Santos-Silva, Alessandra; Guimond, Scott E; Turnbull, Jeremy E; Barnett, Susan C

    2012-11-07

    Previously, it has been shown that rat Schwann cells (SCs), but not olfactory ensheathing cells (OECs), form a boundary with astrocytes, due to a SC-specific secreted factor. Here, we identify highly sulfated heparan sulfates (HSs) and fibroblast growth factors (FGFs) 1 and 9 as possible determinants of boundary formation induced by rat SCs. Disaccharide analysis of HS in SC-conditioned and rat OEC-conditioned media showed that SCs secrete more highly sulfated HS than OECs. The dependence of the boundary-forming activity on high levels of sulfation was confirmed using a panel of semisynthetic modified heparins with variable levels of sulfation. Furthermore, extracellular HS 6-O-endosulfatase enzymes, Sulf 1 and Sulf 2, were expressed at a significantly lower level by SCs compared with OECs, and siRNA reduction of Sulfs in OECs was, in itself, sufficient to induce boundary formation. This demonstrates a key role for remodelling (reduction) of HS 6-O-sulfation by OECs, compared with SCs, to suppress boundary formation. Furthermore, specific anti-FGF1 and anti-FGF9 antibodies disrupted SC-astrocyte boundary formation, supporting a role for an HS sulfation-dependent FGF signaling mechanism via FGF receptors on astrocytes. We propose a model in which FGF1 and FGF9 signaling is differentially modulated by patterns of glial cell HS sulfation, dependent on Sulf 1 and Sulf 2 expression, to control FGF receptor 3-IIIb-mediated astrocytic responses. Moreover, these data suggest manipulation of HS sulfation after CNS injury as a potential novel approach for therapeutic intervention in CNS repair.

  7. The effects of lighting conditions on responses of cells selective for face views in the macaque temporal cortex.

    PubMed

    Hietanen, J K; Perrett, D I; Oram, M W; Benson, P J; Dittrich, W H

    1992-01-01

    Neural mechanisms underlying recognition of objects must overcome the changes in an object's appearance caused by inconsistent viewing conditions, particularly those that occur with changes in lighting. In humans, lesions to the posterior visual association cortex can impair the ability to recognize objects and faces across different lighting conditions. Inferotemporal lesions in monkey have been shown to produce a similar difficulty in object matching tasks. Here we report on the extent to which cell responses selective for the face and other views of the head in monkey temporal cortex tolerate changes in lighting. For each cell studied the (preferred) head view eliciting maximal response was first established under normal lighting. Cells were then tested with the preferred head view lit from different directions (i.e. front, above, below or from the side). Responses of some cells failed to show complete generalization across all lighting conditions but together as a "population" they responded equally strongly under all four lighting conditions. Further tests on sub-groups of cells revealed that stimulus selectivity was maintained despite unusual lighting. The cells discriminated between head and control stimuli and between different views of the head independent of the lighting direction. The results indicate that constancy of recognition across different lighting conditions is apparent in the responses of single cells in the temporal cortex. Lighting constancy appears to be established by matching the retinal image to view-specific descriptions of objects (i.e. neurons which compute object structure from a limited range of perspective views).

  8. Role of reduced intensity conditioning in T-cell and B-cell immune reconstitution after HLA-identical bone marrow transplantation in ADA-SCID.

    PubMed

    Cancrini, Caterina; Ferrua, Francesca; Scarselli, Alessia; Brigida, Immacolata; Romiti, Maria Luisa; Barera, Graziano; Finocchi, Andrea; Roncarolo, Maria Grazia; Caniglia, Maurizio; Aiuti, Alessandro

    2010-10-01

    The treatment of choice for severe combined immunodeficiency is bone marrow transplantation from an HLA-identical donor sibling without conditioning. However, this may result in low donor stem cell chimerism, leading to reduced long-term immune reconstitution. We compared engraftment, metabolic, and T-cell and B-cell immune reconstitution of HLA-identical sibling bone marrow transplantation performed in 2 severe combined immunodeficiency infants with adenosine deaminase deficiency from the same family treated with or without a reduced intensity conditioning regimen (busulfan/fludarabine). Only the patient who received conditioning showed a stable mixed chimerism in all lineages, including bone marrow myeloid and B cells. The use of conditioning resulted in higher thymus-derived naïve T cells and T-cell receptor excision circles, normalization of the T-cell repertoire, and faster and complete B-cell and metabolic reconstitution. These results suggest the utility of exploring the use of reduced intensity conditioning in bone marrow transplantation from HLA-identical donor in severe combined immunodeficiency to improve long-term immune reconstitution.

  9. Paradoxical early glucocorticoid induction of stem cell factor (SCF) expression in inflammatory conditions

    PubMed Central

    Da Silva, Carla Alexandra; Kassel, Olivier; Lebouquin, Renaud; Lacroix, Emmanuel Jean; Frossard, Nelly

    2003-01-01

    Stem cell factor (SCF) is a major growth factor for mast cells, promoting their differentiation and chemotaxis. Its expression is regulated by glucocorticoids in inflammatory conditions, showing an early increased protein expression, before the expected anti-inflammatory decrease (Da Silva et al., Br. J. Pharmacol. 2002:135,1634). We here evaluated the early kinetic of SCF expression regulated by interleukin (IL)-1β, budesonide and the combination of both in human lung fibroblasts in culture. Budesonide potentiated the IL-1β-enhanced expression of SCF mRNA (+103%) and protein (+98%) very shortly after treatment (at 30 min and 1 h, respectively). A gentle downregulation followed. This potentiating effect of budesonide was related to increased SCF mRNA stability and SCF gene transcription. Deletion of a κB-like site that we identified in the first intron of the SCF gene, in a luciferase reporter system, abolished the potentiation by budesonide, as well as the effect of IL-1β alone, as compared to the wild-type construction activity. All budesonide-induced effects were glucocorticoid-receptor dependent, since they were reproduced by dexamethasone and blocked by RU486. IL-1β+budesonide did not affect the relative expression of the soluble and membrane-bound forms of SCF. In conclusion, our results clearly show that glucocorticoids act very early to adversely increase the expression of SCF mRNA and protein in the inflammatory conditions created by IL-1β, and that this effect involves increased mRNA stability and increased gene expression through activation of the NF-κB-like responsive element. PMID:14662725

  10. The biofilm environment offers a possible condition for inducing the competency of DNA recipient cells through nutritional starvation.

    PubMed

    Nishioka, Motomu; Mashayekhan, Shohreh; Onishi, Kyoko; Taya, Masahito

    2007-09-01

    Transformation phenomena occurring under conditions mimicking the biofilm environment were investigated using Escherichia coli IM302 (as DNA recipient cells) and Providencia sp. WW2 (as surrounding cells in the biofilm model). In the case of planktonic IM302 cells kept at 25 degrees C, the transformation took place exclusively in the absence of organic nutrients (COD = 0), and was not substantially observed in the range of COD = 30-1500 mg O2/L. On the other hand, in the case of biofilm IM302 cells, the transformation occurred at relatively high levels under the examined conditions (temperature = 5 or 25 degrees C and COD = 0-1500 mg O2/L). These results indicated that the competency of biofilm IM302 cells was induced even in the presence of organic nutrients owing to nutritional starvation caused by WW2 cells.

  11. Exploring the Limits of Cell Adhesion under Shear Stress within Physiological Conditions and beyond on a Chip.

    PubMed

    Stamp, Melanie E M; Jötten, Anna M; Kudella, Patrick W; Breyer, Dominik; Strobl, Florian G; Geislinger, Thomas M; Wixforth, Achim; Westerhausen, Christoph

    2016-10-21

    Cell adhesion processes are of ubiquitous importance for biomedical applications such as optimization of implant materials. Here, not only physiological conditions such as temperature or pH, but also topographical structures play crucial roles, as inflammatory reactions after surgery can diminish osseointegration. In this study, we systematically investigate cell adhesion under static, dynamic and physiologically relevant conditions employing a lab-on-a-chip system. We screen adhesion of the bone osteosarcoma cell line SaOs-2 on a titanium implant material for pH and temperature values in the physiological range and beyond, to explore the limits of cell adhesion, e.g., for feverish and acidic conditions. A detailed study of different surface roughness Rq gives insight into the correlation between the cells' abilities to adhere and withstand shear flow and the topography of the substrates, finding a local optimum at Rq = 22 nm. We use shear stress induced by acoustic streaming to determine a measure for the ability of cell adhesion under an external force for various conditions. We find an optimum of cell adhesion for T = 37 °C and pH = 7.4 with decreasing cell adhesion outside the physiological range, especially for high T and low pH. We find constant detachment rates in the physiological regime, but this behavior tends to collapse at the limits of 41 °C and pH 4.

  12. The effects of mine conditions on the performance of a PEM fuel cell

    NASA Astrophysics Data System (ADS)

    Bétournay, Marc C.; Bonnell, Gary; Edwardson, Eric; Paktunc, Dogan; Kaufman, Arthur; Lomma, A. Timothy

    Proton exchange membrane fuel cells (PEMFC) have been selected to replace conventional underground power sources such as diesel engines, to improve underground air quality, to reduce green house gas emissions and operating costs and to facilitate equipment automation. The effects of underground mining conditions, gases, dust and shock and vibration on the performance of PEMFC's were investigated during extensive testing in an operating underground metal mine. Neither the voltage-amperage nor the power-amperage curves showed significant damage effects, and a post-testing stack inspection showed minor pressure drop, at the higher current density and airflow rate. With the use of an air intake filter, little particle accumulation was registered in the stack, and effluent water testing revealed the presence of rock-derived particles, showing that the stack was able to purge itself of low particle concentrations. No physical damage was imposed to the stack, auxiliary system and hydrogen metal hydride storage unit. Fuel cell performance compared well to pre-test and initial construction power plant data generation. Further tests are recommended to study individual mine gas and particle mineralogy type effects.

  13. Derivation Conditions Determine X-Reactivation Status in Female Human Induced Pluripotent Stem Cells

    PubMed Central

    Tomoda, Kiichiro; Takahashi, Kazutoshi; Leung, Karen; Okada, Aki; Narita, Megumi; Yamada, N. Alice; Eilertson, Kirsten E.; Tsang, Peter; Baba, Shiro; White, Mark P.; Sami, Salma; Srivastava, Deepak; Conklin, Bruce R.; Panning, Barbara; Yamanaka, Shinya

    2012-01-01

    SUMMARY Female human induced pluripotent stem cell (hiPSC) lines exhibit variability in X-inactivation status. The majority of hiPSC lines maintain one transcriptionally active X (Xa) and one inactive X (Xi) chromosome from donor cells. However, at low frequency, hiPSC lines with two Xas are produced, suggesting that epigenetic alterations of the Xi occur sporadically during reprogramming. We show here that X-inactivation status in female hiPSC lines depends on derivation conditions. hiPSC lines generated by the Kyoto method (retroviral or episomal reprogramming), which uses leukemia inhibitory factor (LIF)-expressing SNL feeders, frequently had two Xas. Early passage Xa/Xi hiPSC lines generated on non-SNL feeders were converted into Xa/Xa hiPSC lines after several passages on SNL feeders, and supplementation with recombinant LIF caused reactivation of some of X-linked genes. Thus, feeders are a significant factor affecting X-inactivation status. The efficient production of Xa/Xa hiPSC lines provides unprecedented opportunities to understand human X-reactivation and inactivation. PMID:22770243

  14. Antioxidant and neuronal cell protective effects of columbia arabica coffee with different roasting conditions.

    PubMed

    Jeong, Ji Hee; Jeong, Hee Rok; Jo, Yu Na; Kim, Hyun Ju; Lee, Uk; Heo, Ho Jin

    2013-03-01

    In vitro antioxidant activities and neuronal cell protective effects of ethanol extract from roasted coffee beans were investigated. Colombia arabica coffee (Coffea arabica) green beans were roasted to give medium (230°C, 10 min), city (230°C, 12 min) and french (230°C, 15 min) coffee beans. Total phenolics in raw green beans, medium, city and french-roasted beans were 8.81±0.05, 9.77±0.03, 9.92±0.04 and 7.76±0.01 mg of GAE/g, respectively. The content of 5-O-caffeoylquinic acid, the predominant phenolic, was detected higher in medium-roasted beans than others. In addition, we found that extracts from medium-roasted beans particularly showed the highest in vitro antioxidant activity on ABTS radical scavenging activity and FRAP assays. To determine cell viability using the MTT assay, extracts from medium-roasted beans showed higher protection against H2O2-induced neurotoxicity than others. Lactate dehydrogenase (LDH) leakage was also inhibited by the extracts due to prevention of lipid peroxidation using the malondialdehyde (MDA) assay from mouse whole brain homogenates. These data suggest that the medium-roasting condition to making tasty coffee from Columbia arabica green beans may be more helpful to human health by providing the most physiological phenolics, including 5-O-caffeoylquinic acids.

  15. A homologue of cathepsin L identified in conditioned medium from Sf9 insect cells.

    PubMed

    Lindskog, Eva; Svensson, Ingrid; Häggström, Lena

    2006-07-01

    Gelatin zymography revealed the presence of proteolytic activity in conditioned medium (CM) from a serum-free, non-infected Spodoptera frugiperda, Sf9 insect cell culture. Two peptidase bands at about 49 and 39 kDa were detected and found to be proform and active form of the same enzyme. The 49-kDa form was visible on zymogram gels in samples of CM taken on days 4 and 5 of an Sf9 culture, while the 39-kDa form was seen on days 6 and 7. On basis of the inhibitor profile and substrate range, the enzyme was identified as an Sf9 homologue of cathepsin L, a papain-like cysteine peptidase. After lowering the pH of Sf9 CM to 3.5, an additional peptidase band at 22 kDa appeared. This peptidase showed the same inhibitor profile, substrate range and optimum pH (5.0) as the 39-kDa form, indicating that Sf9 cathepsin L has two active forms, at 39 and 22 kDa. Addition of the cysteine peptidase inhibitor E-64c to an Sf9 culture inhibited all proteolytic activities of Sf9 cathepsin L but did not influence the proliferation of Sf9 cells.

  16. Anti-inflammatory effect of conditioned medium from human uterine cervical stem cells in uveitis.

    PubMed

    Bermudez, Maria A; Sendon-Lago, Juan; Seoane, Samuel; Eiro, Noemi; Gonzalez, Francisco; Saa, Jorge; Vizoso, Francisco; Perez-Fernandez, Roman

    2016-08-01

    The aim of the present study was to evaluate the effect of conditioned medium from human uterine cervical stem cells (CM-hUCESCs) in uveitis. To do that, uveitis was induced in rats after footpad injection of Escherichia coli lipopolysaccaride (LPS). Human retinal pigment epithelial (ARPE-19) cells after LPS challenge were used to test anti-inflammatory effect of CM-hUCESCs 'ìn vitro'. Real-time PCR was used to evaluate mRNA expression levels of the pro-inflammatory cytokines interkeukin-6, interkeukin-8, macrophage inflammatory protein-1 alpha, tumor necrosis factor alpha, and the anti-inflammatory interkeukin-10. Leucocytes from aqueous humor (AqH) were quantified in a Neubauer chamber, and eye histopathological analysis was done with hematoxylin-eosin staining. Additionally, using a human cytokine antibody array we evaluated CM-hUCESCs to determine mediating proteins. Results showed that administration of CM-hUCESCs significantly reduced LPS-induced pro-inflammatory cytokines both 'in vitro' and 'in vivo', and decreased leucocytes in AqH and ocular tissues. High levels of cytokines with anti-inflammatory effects were found in CM-hUCESCs, suggesting a possible role of these factors in reducing intraocular inflammation. In summary, treatment with CM-hUCESCs significantly reduces inflammation in uveitis. Our data indicate that CM-hUCESCs could be regarded as a potential therapeutic agent for patients suffering from ocular inflammation.

  17. Classification of yeast cells from image features to evaluate pathogen conditions

    NASA Astrophysics Data System (ADS)

    van der Putten, Peter; Bertens, Laura; Liu, Jinshuo; Hagen, Ferry; Boekhout, Teun; Verbeek, Fons J.

    2007-01-01

    Morphometrics from images, image analysis, may reveal differences between classes of objects present in the images. We have performed an image-features-based classification for the pathogenic yeast Cryptococcus neoformans. Building and analyzing image collections from the yeast under different environmental or genetic conditions may help to diagnose a new "unseen" situation. Diagnosis here means that retrieval of the relevant information from the image collection is at hand each time a new "sample" is presented. The basidiomycetous yeast Cryptococcus neoformans can cause infections such as meningitis or pneumonia. The presence of an extra-cellular capsule is known to be related to virulence. This paper reports on the approach towards developing classifiers for detecting potentially more or less virulent cells in a sample, i.e. an image, by using a range of features derived from the shape or density distribution. The classifier can henceforth be used for automating screening and annotating existing image collections. In addition we will present our methods for creating samples, collecting images, image preprocessing, identifying "yeast cells" and creating feature extraction from the images. We compare various expertise based and fully automated methods of feature selection and benchmark a range of classification algorithms and illustrate successful application to this particular domain.

  18. Characterization and optimization of cathodic conditions for H2O2 synthesis in microbial electrochemical cells.

    PubMed

    Sim, Junyoung; An, Junyeong; Elbeshbishy, Elsayed; Ryu, Hodon; Lee, Hyung-Sool

    2015-11-01

    Cathode potential and O2 supply methods were investigated to improve H2O2 synthesis in an electrochemical cell, and optimal cathode conditions were applied for microbial electrochemical cells (MECs). Using aqueous O2 for the cathode significantly improved current density, but H2O2 conversion efficiency was negligible at 0.3-12%. Current density decreased for passive O2 diffusion to the cathode, but H2O2 conversion efficiency increased by 65%. An MEC equipped with a gas diffusion cathode was operated with acetate medium and domestic wastewater, which presented relatively high H2O2 conversion efficiency from 36% to 47%, although cathode overpotential was fluctuated. Due to different current densities, the maximum H2O2 production rate was 141 mg H2O2/L-h in the MEC fed with acetate medium, but it became low at 6 mg H2O2/L-h in the MEC fed with the wastewater. Our study clearly indicates that improving anodic current density and mitigating membrane fouling would be key parameters for large-scale H2O2-MECs.

  19. Optimizing operating conditions and electrochemical characterization of glucose-gluconate alkaline fuel cells

    NASA Astrophysics Data System (ADS)

    Pasta, M.; La Mantia, F.; Ruffo, R.; Peri, F.; Pina, C. Della; Mari, C. M.

    The direct oxidation of glucose to produce electrical energy has been widely investigated because of renewability, abundance, high energy density and easy handling of the carbohydrate. Most of the previous studies have been conducted in extreme conditions in order to achieve complete glucose oxidation to CO 2, neglecting the carbohydrate chemical instability that generally leads to useless by-products mixtures. The partial oxidation to gluconate, originally studied for implantable fuel cells, has the advantage of generating a commercially valuable chemical. In the present paper we optimized fuel composition and operating conditions in order to selectively oxidize glucose to gluconate, maximizing the power density output of a standard commercial platinum based anode material. A deep electrochemical characterization concerning reversible potential, cyclic voltammetry and overpotential measurements have been carried out at 25 °C in the D-(+)-glucose concentration range 1.0 × 10 -2 to 1.0 M. NMR and EIS investigation clarify the role of the buffer in enhancing the electrochemical performance.

  20. Ovarian Function after Hematopoietic Cell Transplantation: A Descriptive Study Following the Use of GnRH Agonists for Myeloablative Conditioning and Observation Only for Reduced-Intensity Conditioning

    PubMed Central

    Phelan, Rachel; Mann, Elizabeth; Napurski, Char; DeFor, Todd E; Petryk, Anna; Miller, Weston P; Wagner, John E; Verneris, Michael R; Smith, Angela R

    2017-01-01

    Gonadal failure is a health and quality of life concern in hematopoietic cell transplant (HCT) survivors. While ovarian dysfunction is nearly universal following myeloablative (MA) conditioning, risk is unclear after reduced-intensity conditioning (RIC). Gonadotropin-releasing hormone agonists decrease ovarian failure rates following conventional chemotherapy but little is known about its effectiveness with HCT. We investigated the impact of leuprolide on ovarian function after MA conditioning and monitored ovarian function after RIC in this descriptive pilot study. Post-menarchal females <50 years undergoing HCT with adequate baseline ovarian function (FSH level <40 mIU/mL and normal menstruation) were eligible. Prior to MA conditioning, leuprolide was administered. Those undergoing RIC were observed. FSH was measured at various time points. Seventeen women aged 12–45 years were evaluated (7 in the intervention group and 10 observation group). Compared to the historical high rate of ovarian failure after MA conditioning, 3 of 7 evaluable Lupron recipients had ovarian failure at a median of 703 days post-transplant. Ovarian failure occurred in 1 of 10 recipients of RIC at median follow-up of 901 days. In conclusion, leuprolide may protect ovarian function after MA conditioning. Additionally, RIC with cyclophosphamide, fludarabine and low-dose TBI has a low risk of ovarian failure. PMID:27272448

  1. Osteoblast-conditioned media influence the expression of E-selectin on bone-derived vascular endothelial cells.

    PubMed

    Makuch, Lauren A; Sosnoski, Donna M; Gay, Carol V

    2006-08-01

    Breast cancer cells frequently metastasize to the ends of long bones, ribs and vertebrae, structures which contain a rich microvasculature that is closely juxtaposed to metabolically active trabecular bone surfaces. This study focuses on the effects of osteoblast secretions on the surface presentation of adhesive proteins on skeletal vascular endothelial cells. Vascular endothelial cells were isolated from trabecular bone regions of the long bones of 7-week-old Swiss Webster mice and also from the central marrow cavity where trabecular bone is absent. Both types of endothelial cells were placed in culture for 7 days, then exposed 24 h to conditioned media from MC3T3-E1 osteoblasts. Conditioned medium (CM) from two different stages of osteoblast development were tested: (1) from immature MC3T3-E1 cells cultured for 5-7 days and (2) from mature MC3T3-E1 cells cultured for 28-30 days. The immature osteoblasts were in a stage of rapid proliferation; the mature osteoblasts formed a matrix that mineralized. Following exposure to the conditioned media, the vascular cells were exposed to anti-P-selectin, anti-E-selectin, anti-ICAM-1, and anti-VCAM-1 to detect the corresponding adhesive proteins on their surfaces. Breast cancer cells are known to bind to these adhesive proteins. Of the four proteins evaluated, E-selectin was consistently found on more cell surfaces (approximately 30%) of bone-derived vascular endothelial cells (BVECs) when exposed to the immature CM whereas vascular endothelial cells from marrow (MVECs) did not show this response to either immature CM or mature CM. These studies suggest that the BVEC blood vessels near immature bone cells express more surface adhesive protein that could enhance entrapment and extravasation of breast cancer cells. Once cancer cells have undergone extravasation into marrow adjacent to bone, they could be readily attracted to nearby bone surfaces.

  2. A conditional transgenic mouse line for targeted expression of the stem cell marker LGR5.

    PubMed

    Norum, Jens Henrik; Bergström, Åsa; Andersson, Agneta Birgitta; Kuiper, Raoul V; Hoelzl, Maria A; Sørlie, Therese; Toftgård, Rune

    2015-08-15

    LGR5 is a known marker of embryonic and adult stem cells in several tissues. In a mouse model, Lgr5+ cells have shown tumour-initiating properties, while in human cancers, such as basal cell carcinoma and colon cancer, LGR5 expression levels are increased: however, the effect of increased LGR5 expression is not fully understood. To study the effects of elevated LGR5 expression levels we generated a novel tetracycline-responsive, conditional transgenic mouse line expressing human LGR5, designated TRELGR5. In this transgenic line, LGR5 expression can be induced in any tissue depending on the expression pattern of the chosen transcriptional regulator. For the current study, we used transgenic mice with a tetracycline-regulated transcriptional transactivator linked to the bovine keratin 5 promoter (K5tTA) to drive expression of LGR5 in the epidermis. As expected, expression of human LGR5 was induced in the skin of double transgenic mice (K5tTA;TRELGR5). Inducing LGR5 expression during embryogenesis and early development resulted in macroscopically and microscopically detectable phenotypic changes, including kink tail, sparse fur coat and enlarged sebaceous glands. The fur and sebaceous gland phenotypes were reversible upon discontinued expression of transgenic LGR5, but this was not observed for the kink tail phenotype. There were no apparent phenotypic changes if LGR5 expression was induced at three weeks of age. The results demonstrate that increased expression of LGR5 during embryogenesis and the neonatal period alter skin development and homeostasis.

  3. Modeling of Thermal Performance of Multiphase Nuclear Fuel Cell Under Variable Gravity Conditions

    NASA Technical Reports Server (NTRS)

    Ding, Z.; Anghaie, S.

    1996-01-01

    A unique numerical method has been developed to model the dynamic processes of bulk evaporation and condensation processes, associated with internal heat generation and natural convection under different gravity levels. The internal energy formulation, for the bulk liquid-vapor phase change problems in an encapsulated container, was employed. The equations, governing the conservation of mass, momentum and energy for both phases involved in phase change, were solved. The thermal performance of a multiphase uranium tetra-fluoride fuel element under zero gravity, micro-gravity and normal gravity conditions has been investigated. The modeling yielded results including the evolution of the bulk liquid-vapor phase change process, the evolution of the liquid-vapor interface, the formation and development of the liquid film covering the side wall surface, the temperature distribution and the convection flow field in the fuel element. The strong dependence of the thermal performance of such multiphase nuclear fuel cell on the gravity condition has been revealed. Under all three gravity conditions, 0-g, 10(exp -3)-g, and 1-g, the liquid film is formed and covers the entire side wall. The liquid film covering the side wall is more isothermalized at the wall surface, which can prevent the side wall from being over-heated. As the gravity increases, the liquid film is thinner, the temperature gradient is larger across the liquid film and smaller across the vapor phase. This investigation provides valuable information about the thermal performance of multi-phase nuclear fuel element for the potential space and ground applications.

  4. Memory-like CD8+ and CD4+ T cells cooperate to break peripheral tolerance under lymphopenic conditions.

    PubMed

    Le Saout, Cecile; Mennechet, Sandie; Taylor, Naomi; Hernandez, Javier

    2008-12-09

    The onset of autoimmunity in experimental rodent models and patients frequently correlates with a lymphopenic state. In this condition, the immune system has evolved compensatory homeostatic mechanisms that induce quiescent naive T cells to proliferate and differentiate into memory-like lymphocytes even in the apparent absence of antigenic stimulation. Because memory T cells have less stringent requirements for activation than naive cells, we hypothesized that autoreactive T cells that arrive to secondary lymphoid organs in a lymphopenic environment could differentiate and bypass the mechanisms of peripheral tolerance such as those mediated by self-antigen cross-presentation. Here, we show that lymphopenia-driven proliferation and differentiation of potentially autoreactive CD8(+) T cells into memory-like cells is not sufficient to induce self-reactivity against a pancreatic antigen. Induction of an organ-specific autoimmunity required antigen-specific CD4(+) T cell help. Notably, we found that this function could be accomplished by memory-like CD4(+) T cells generated in vivo through lymphopenia-induced proliferation. These helper cells promoted the further differentiation of memory-like CD8(+) T cells into effectors in response to antigen cross-presentation, resulting in their migration to the tissue of antigen expression where autoimmunity ensued. Thus, the cooperation of self-reactive memory-like CD4(+) and CD8(+) T cells under lymphopenic conditions overcomes cross-tolerance resulting in autoimmunity.

  5. Effect of sevoflurane on human hepatocellular carcinoma HepG2 cells under conditions of high glucose and insulin.

    PubMed

    Nishiwada, Tadashi; Kawaraguchi, Yoshitaka; Uemura, Keiko; Sugimoto, Hiroshi; Kawaguchi, Masahiko

    2015-10-01

    Diabetes mellitus is associated with morbidity and progression of some cancers, such as hepatocellular carcinoma. It has been reported that sevoflurane, a volatile anesthetic agent commonly used in cancer surgery, can lead to lower overall survival rates than those observed when propofol is used to treat cancer patients, and sevoflurane increases cancer cell proliferation in in vitro studies. It has been also reported that glucose levels in rats anesthetized with sevoflurane were higher than those in rats anesthetized with propofol. We investigated the effect of sevoflurane, under conditions of high glucose and insulin, on cell proliferation in the human hepatocellular carcinoma cell line, HepG2. First, we exposed HepG2 cells to sevoflurane at 1 or 2 % concentration for 6 h in various glucose concentrations and then evaluated cell proliferation using the MTT assay. Subsequently, to mimic diabetic conditions observed during surgery, HepG2 cells were exposed to sevoflurane at 1 or 2 % concentration in high glucose concentrations at various concentrations of insulin for 6 h. One-percent sevoflurane exposure enhanced cell proliferation under conditions of high glucose, treated with 0.05 mg/l insulin. Our study implies that sevoflurane may affect cell proliferation in human hepatocellular carcinoma cells in a physiological situation mimicking that of diabetes.

  6. AN INVESTIGATION TO RESOLVE THE INTERACTION BETWEEN FUEL CELL, POWER CONDITIONING SYSTEM AND APPLICATION LOADS

    SciTech Connect

    Sudip K. Mazumder; Chuck McKintyre; Dan Herbison; Doug Nelson; Comas Haynes; Michael von Spakovsky; Joseph Hartvigsen; S. Elangovan

    2003-11-03

    Solid-Oxide Fuel Cell (SOFC) stacks respond quickly to changes in load and exhibit high part- and full-load efficiencies due to its rapid electrochemistry. However, this is not true for the thermal, mechanical, and chemical balance-of-plant subsystem (BOPS), where load-following time constants are, typically, several orders of magnitude higher. This dichotomy diminishes the reliability and performance of the electrode with increasing demand of load. Because these unwanted phenomena are not well understood, the manufacturers of SOFC use conservative schemes (such as, delayed load-following to compensate for slow BOPS response or expensive inductor filtering) to control stack responses to load variations. This limits the applicability of SOFC systems for load-varying stationary and transportation applications from a cost standpoint. Thus, a need exists for the synthesis of component- and system-level models of SOFC power-conditioning systems and the development of methodologies for investigating the system-interaction issues (which reduce the lifetime and efficiency of a SOFC) and optimizing the responses of each subsystem, leading to optimal designs of power-conditioning electronics and optimal control strategies, which mitigate the electrical-feedback effects. Equally important are ''multiresolution'' finite-element modeling and simulation studies, which can predict the impact of changes in system-level variables (e.g., current ripple and load-transients) on the local current densities, voltages, and temperature (these parameters are very difficult or cumbersome, if not impossible to obtain) within a SOFC cell. Towards that end, for phase I of this project, sponsored by the U.S. DOE (NETL), we investigate the interactions among fuel cell, power-conditioning system, and application loads and their effects on SOFC reliability (durability) and performance. A number of methodologies have been used in Phase I to develop the steady-state and transient nonlinear models of

  7. Strigolactone analogues induce apoptosis through activation of p38 and the stress response pathway in cancer cell lines and in conditionally reprogrammed primary prostate cancer cells.

    PubMed

    Pollock, Claire B; McDonough, Sara; Wang, Victor S; Lee, Hyojung; Ringer, Lymor; Li, Xin; Prandi, Cristina; Lee, Richard J; Feldman, Adam S; Koltai, Hinanit; Kapulnik, Yoram; Rodriguez, Olga C; Schlegel, Richard; Albanese, Christopher; Yarden, Ronit I

    2014-03-30

    Strigolactones are a novel class of plant hormones produced in roots and regulate shoot and root development. We have previously shown that synthetic strigolactone analogues potently inhibit growth of breast cancer cells and breast cancer stem cells. Here we show that strigolactone analogues inhibit the growth and survival of an array of cancer-derived cell lines representing solid and non-solid cancer cells including: prostate, colon, lung, melanoma, osteosarcoma and leukemic cell lines, while normal cells were minimally affected. Treatment of cancer cells with strigolactone analogues was hallmarked by activation of the stress-related MAPKs: p38 and JNK and induction of stress-related genes; cell cycle arrest and apoptosis evident by increased percentages of cells in the sub-G1 fraction and Annexin V staining. In addition, we tested the response of patient-matched conditionally reprogrammed primary prostate normal and cancer cells. The tumor cells exhibited significantly higher sensitivity to the two most potent SL analogues with increased apoptosis confirmed by PARP1 cleavage compared to their normal counterpart cells. Thus, Strigolactone analogues are promising candidates for anticancer therapy by their ability to specifically induce cell cycle arrest, cellular stress and apoptosis in tumor cells with minimal effects on growth and survival of normal cells.

  8. Stem cell conditioned medium improves acute lung injury in mice: in vivo evidence for stem cell paracrine action

    PubMed Central

    Ionescu, Lavinia; Byrne, Roisin N.; van Haaften, Tim; Vadivel, Arul; Alphonse, Rajesh S.; Rey-Parra, Gloria J.; Weissmann, Gaia; Hall, Adam; Eaton, Farah

    2012-01-01

    Mortality and morbidity of acute lung injury and acute respiratory distress syndrome remain high because of the lack of pharmacological therapies to prevent injury or promote repair. Mesenchymal stem cells (MSCs) prevent lung injury in various experimental models, despite a low proportion of donor-derived cell engraftment, suggesting that MSCs exert their beneficial effects via paracrine mechanisms. We hypothesized that soluble factors secreted by MSCs promote the resolution of lung injury in part by modulating alveolar macrophage (AM) function. We tested the therapeutic effect of MSC-derived conditioned medium (CdM) compared with whole MSCs, lung fibroblasts, and fibroblast-CdM. Intratracheal MSCs and MSC-CdM significantly attenuated lipopolysaccharide (LPS)-induced lung neutrophil influx, lung edema, and lung injury as assessed by an established lung injury score. MSC-CdM increased arginase-1 activity and Ym1 expression in LPS-exposed AMs. In vivo, AMs from LPS-MSC and LPS-MSC CdM lungs had enhanced expression of Ym1 and decreased expression of inducible nitric oxide synthase compared with untreated LPS mice. This suggests that MSC-CdM promotes alternative macrophage activation to an M2 “healer” phenotype. Comparative multiplex analysis of MSC- and fibroblast-CdM demonstrated that MSC-CdM contained several factors that may confer therapeutic benefit, including insulin-like growth factor I (IGF-I). Recombinant IGF-I partially reproduced the lung protective effect of MSC-CdM. In summary, MSCs act through a paracrine activity. MSC-CdM promotes the resolution of LPS-induced lung injury by attenuating lung inflammation and promoting a wound healing/anti-inflammatory M2 macrophage phenotype in part via IGF-I. PMID:23023971

  9. Relation between cell death progression, reactive oxygen species production and mitochondrial membrane potential in fermenting Saccharomyces cerevisiae cells under heat-shock conditions.

    PubMed

    Pyatrikas, Darya V; Fedoseeva, Irina V; Varakina, Nina N; Rusaleva, Tatyana M; Stepanov, Alexei V; Fedyaeva, Anna V; Borovskii, Gennadii B; Rikhvanov, Eugene G

    2015-06-01

    Moderate heat shock increased reactive oxygen species (ROS) production that led to cell death in glucose-grown Saccharomyces cerevisiae cells. Conditions that disturb mitochondrial functions such as treatment by uncouplers and petite mutation were shown to inhibit ROS production and protects cell from thermal death. Hence, mitochondria are responsible for ROS production and play an active role in cell death. An increase in ROS production was accompanied by hyperpolarization of inner mitochondrial membrane. All agents suppressing hyperpolarization also suppressed heat-induced ROS production. It was supposed that generation of ROS under moderate heat shock in glucose-grown S. cerevisiae cells is driven by the mitochondrial membrane potential.

  10. Exploring the Limits of Cell Adhesion under Shear Stress within Physiological Conditions and beyond on a Chip

    PubMed Central

    Stamp, Melanie E. M.; Jötten, Anna M.; Kudella, Patrick W.; Breyer, Dominik; Strobl, Florian G.; Geislinger, Thomas M.; Wixforth, Achim; Westerhausen, Christoph

    2016-01-01

    Cell adhesion processes are of ubiquitous importance for biomedical applications such as optimization of implant materials. Here, not only physiological conditions such as temperature or pH, but also topographical structures play crucial roles, as inflammatory reactions after surgery can diminish osseointegration. In this study, we systematically investigate cell adhesion under static, dynamic and physiologically relevant conditions employing a lab-on-a-chip system. We screen adhesion of the bone osteosarcoma cell line SaOs-2 on a titanium implant material for pH and temperature values in the physiological range and beyond, to explore the limits of cell adhesion, e.g., for feverish and acidic conditions. A detailed study of different surface roughness Rq gives insight into the correlation between the cells’ abilities to adhere and withstand shear flow and the topography of the substrates, finding a local optimum at Rq = 22 nm. We use shear stress induced by acoustic streaming to determine a measure for the ability of cell adhesion under an external force for various conditions. We find an optimum of cell adhesion for T = 37 °C and pH = 7.4 with decreasing cell adhesion outside the physiological range, especially for high T and low pH. We find constant detachment rates in the physiological regime, but this behavior tends to collapse at the limits of 41 °C and pH 4. PMID:27775638

  11. Human immune cells' behavior and survival under bioenergetically restricted conditions in an in vitro fracture hematoma model.

    PubMed

    Hoff, Paula; Maschmeyer, Patrick; Gaber, Timo; Schütze, Tabea; Raue, Tobias; Schmidt-Bleek, Katharina; Dziurla, René; Schellmann, Saskia; Lohanatha, Ferenz Leonard; Röhner, Eric; Ode, Andrea; Burmester, Gerd-Rüdiger; Duda, Georg N; Perka, Carsten; Buttgereit, Frank

    2013-03-01

    The initial inflammatory phase of bone fracture healing represents a critical step for the outcome of the healing process. However, both the mechanisms initiating this inflammatory phase and the function of immune cells present at the fracture site are poorly understood. In order to study the early events within a fracture hematoma, we established an in vitro fracture hematoma model: we cultured hematomas forming during an osteotomy (artificial bone fracture) of the femur during total hip arthroplasty (THA) in vitro under bioenergetically controlled conditions. This model allowed us to monitor immune cell populations, cell survival and cytokine expression during the early phase following a fracture. Moreover, this model enabled us to change the bioenergetical conditions in order to mimic the in vivo situation, which is assumed to be characterized by hypoxia and restricted amounts of nutrients. Using this model, we found that immune cells adapt to hypoxia via the expression of angiogenic factors, chemoattractants and pro-inflammatory molecules. In addition, combined restriction of oxygen and nutrient supply enhanced the selective survival of lymphocytes in comparison with that of myeloid derived cells (i.e., neutrophils). Of note, non-restricted bioenergetical conditions did not show any similar effects regarding cytokine expression and/or different survival rates of immune cell subsets. In conclusion, we found that the bioenergetical conditions are among the crucial factors inducing the initial inflammatory phase of fracture healing and are thus a critical step for influencing survival and function of immune cells in the early fracture hematoma.

  12. Isolation and expansion of human pluripotent stem cell-derived hepatic progenitor cells by growth factor defined serum-free culture conditions.

    PubMed

    Fukuda, Takayuki; Takayama, Kazuo; Hirata, Mitsuhi; Liu, Yu-Jung; Yanagihara, Kana; Suga, Mika; Mizuguchi, Hiroyuki; Furue, Miho K

    2017-03-15

    Limited growth potential, narrow ranges of sources, and difference in variability and functions from batch to batch of primary hepatocytes cause a problem for predicting drug-induced hepatotoxicity during drug development. Human pluripotent stem cell (hPSC)-derived hepatocyte-like cells in vitro are expected as a tool for predicting drug-induced hepatotoxicity. Several studies have already reported efficient methods for differentiating hPSCs into hepatocyte-like cells, however its differentiation process is time-consuming, labor-intensive, cost-intensive, and unstable. In order to solve this problem, expansion culture for hPSC-derived hepatic progenitor cells, including hepatic stem cells and hepatoblasts which can self-renewal and differentiate into hepatocytes should be valuable as a source of hepatocytes. However, the mechanisms of the expansion of hPSC-derived hepatic progenitor cells are not yet fully understood. In this study, to isolate hPSC-derived hepatic progenitor cells, we tried to develop serum-free growth factor defined culture conditions using defined components. Our culture conditions were able to isolate and grow hPSC-derived hepatic progenitor cells which could differentiate into hepatocyte-like cells through hepatoblast-like cells. We have confirmed that the hepatocyte-like cells prepared by our methods were able to increase gene expression of cytochrome P450 enzymes upon encountering rifampicin, phenobarbital, or omeprazole. The isolation and expansion of hPSC-derived hepatic progenitor cells in defined culture conditions should have advantages in terms of detecting accurate effects of exogenous factors on hepatic lineage differentiation, understanding mechanisms underlying self-renewal ability of hepatic progenitor cells, and stably supplying functional hepatic cells.

  13. Macrolide Antibiotics Exhibit Cytotoxic Effect under Amino Acid-Depleted Culture Condition by Blocking Autophagy Flux in Head and Neck Squamous Cell Carcinoma Cell Lines

    PubMed Central

    Hirasawa, Kazuhiro; Moriya, Shota; Miyahara, Kana; Kazama, Hiromi; Hirota, Ayako; Takemura, Jun; Abe, Akihisa; Inazu, Masato; Hiramoto, Masaki; Tsukahara, Kiyoaki

    2016-01-01

    Autophagy, a self-digestive system for cytoplasmic components, is required to maintain the amino acid pool for cellular homeostasis. We previously reported that the macrolide antibiotics azithromycin (AZM) and clarithromycin (CAM) have an inhibitory effect on autophagy flux, and they potently enhance the cytocidal effect of various anticancer reagents in vitro. This suggests that macrolide antibiotics can be used as an adjuvant for cancer chemotherapy. Since cancer cells require a larger metabolic demand than normal cells because of their exuberant growth, upregulated autophagy in tumor cells has now become the target for cancer therapy. In the present study, we examined whether macrolides exhibit cytotoxic effect under an amino acid-starving condition in head and neck squamous cancer cell lines such as CAL 27 and Detroit 562 as models of solid tumors with an upregulated autophagy in the central region owing to hypovascularity. AZM and CAM induced cell death under the amino acid-depleted (AAD) culture condition in these cell lines along with CHOP upregulation, although they showed no cytotoxicity under the complete culture medium. CHOP knockdown by siRNA in the CAL 27 cells significantly suppressed macrolide-induced cell death under the AAD culture condition. CHOP-/- murine embryonic fibroblast (MEF) cell lines also attenuated AZM-induced cell death compared with CHOP+/+ MEF cell lines. Using a tet-off atg5 MEF cell line, knockout of atg5, an essential gene for autophagy, also induced cell death and CHOP in the AAD culture medium but not in the complete culture medium. This suggest that macrolide-induced cell death via CHOP induction is dependent on autophagy inhibition. The cytotoxicity of macrolide with CHOP induction was completely cancelled by the addition of amino acids in the culture medium, indicating that the cytotoxicity is due to the insufficient amino acid pool. These data suggest the possibility of using macrolides for “tumor-starving therapy”. PMID

  14. A Trial of Alemtuzumab Adjunctive Therapy in Allogeneic Hematopoietic Cell Transplantation with Minimal Conditioning for Severe Combined Immunodeficiency

    PubMed Central

    Dvorak, Christopher C.; Horn, Biljana N.; Puck, Jennifer M.; Adams, Stuart; Veys, Paul; Czechowicz, Agnieszka; Cowan, Morton J.

    2014-01-01

    For infants with severe combined immunodeficiency (SCID) the ideal conditioning regimen before allogeneic hematopoietic cell transplantation (HCT) would omit cytotoxic chemotherapy to minimize short- and long-term complications. We performed a prospective pilot trial with alemtuzumab monotherapy to overcome NK-cell mediated immunologic barriers to engraftment. We enrolled 4 patients who received CD34-selected haploidentical cells, two of whom failed to engraft donor T cells. The 2 patients who engrafted had delayed T cell reconstitution, despite rapid clearance of circulating alemtuzumab. Although well-tolerated, alemtuzumab failed to overcome immunologic barriers to donor engraftment. Furthermore, alemtuzumab may slow T cell development in patients with SCID in the setting of a T-cell depleted graft. PMID:24977928

  15. Examination of various cell culture techniques for co-incubation of virulent Treponema pallidum (Nichols I strain) under anaerobic conditions.

    PubMed Central

    Sandok, P L; Knight, S T; Jenkin, H M

    1976-01-01

    Treponema pallidum (Nichols virulent) was incubated with and without cells in cell culture medium reduced to -275 mV Ecal, pH 7.3, under deoxygenated conditions. Five to ten percent of the treponemes attached to cells and remained motile for at least 120 h in cell-treponeme systems of co-incubation. Virulent treponemes could be detected after 120 to 144 h in the supernatant fluids of cell-treponeme co-incubation cultures and in cell-free tubes containing medium harvested from aerobically cultivated mammalian cells. Medium supplemented with ox serum ultrafiltrate, pyruvate, and sodium thioglycolate and gas mixtures containing H2 and CO2 enhanced treponemal survival. Increases in treponemal numbers were observed using dark-field microscopy but were not substantiated using the rabbit lesion test. Continuous passage of the treponeme was not achieved in vitro. PMID:789395

  16. Optical-cell evidence for superheated ice under gas-hydrate-forming conditions

    USGS Publications Warehouse

    Stern, L.A.; Hogenboom, D.L.; Durham, W.B.; Kirby, S.H.; Chou, I.-Ming

    1998-01-01

    We previously reported indirect but compelling evidence that fine-grained H2O ice under elevated CH4 gas pressure can persist to temperatures well above its ordinary melting point while slowly reacting to form methane clathrate hydrate. This phenomenon has now been visually verified by duplicating these experiments in an optical cell while observing the very slow hydrate-forming process as the reactants were warmed from 250 to 290 K at methane pressures of 23 to 30 MPa. Limited hydrate growth occurred rapidly after initial exposure of the methane gas to the ice grains at temperatures well within the ice subsolidus region. No evidence for continued growth of the hydrate phase was observed until samples were warmed above the equilibrium H2O melting curve. With continued heating, no bulk melting of the ice grains or free liquid water was detected anywhere within the optical cell until hydrate dissociation conditions were reached (292 K at 30 MPa), even though full conversion of the ice grains to hydrate requires 6-8 h at temperatures approaching 290 K. In a separate experimental sequence, unreacted portions of H2O ice grains that had persisted to temperatures above their ordinary melting point were successfully induced to melt, without dissociating the coexisting hydrate in the sample tube, by reducing the pressure overstep of the equilibrium phase boundary and thereby reducing the rate of hydrate growth at the ice-hydrate interface. Results from similar tests using CO2 as the hydrate-forming species demonstrated that this superheating effect is not unique to the CH4-H2O system.

  17. Conditions for diffusion-limited and reaction-limited recombination in nanostructured solar cells

    SciTech Connect

    Ansari-Rad, Mehdi; Anta, Juan A.; Arzi, Ezatollah

    2014-04-07

    The performance of Dye-sensitized solar cells (DSC) and related devices made of nanostructured semiconductors relies on a good charge separation, which in turn is achieved by favoring charge transport against recombination. Although both processes occur at very different time scales, hence ensuring good charge separation, in certain cases the kinetics of transport and recombination can be connected, either in a direct or an indirect way. In this work, the connection between electron transport and recombination in nanostructured solar cells is studied both theoretically and by Monte Carlo simulation. Calculations using the Multiple-Trapping model and a realistic trap distribution for nanostructured TiO{sub 2} show that for attempt-to-jump frequencies higher than 10{sup 11}–10{sup 13} Hz, the system adopts a reaction limited (RL) regime, with a lifetime which is effectively independent from the speed of the electrons in the transport level. For frequencies lower than those, and depending on the concentration of recombination centers in the material, the system enters a diffusion-limited regime (DL), where the lifetime increases if the speed of free electrons decreases. In general, the conditions for RL or DL recombination depend critically on the time scale difference between recombination kinetics and free-electron transport. Hence, if the former is too rapid with respect to the latter, the system is in the DL regime and total thermalization of carriers is not possible. In the opposite situation, a RL regime arises. Numerical data available in the literature, and the behavior of the lifetime with respect to (1) density of recombination centers and (2) probability of recombination at a given center, suggest that a typical DSC in operation stays in the RL regime with complete thermalization, although a transition to the DL regime may occur for electrolytes or hole conductors where recombination is especially rapid or where there is a larger dispersion of energies of

  18. Allogeneic stem cell transplantation using non-myeloablative conditioning regimens: results of the Mexican approach.

    PubMed

    Ruiz-Argüelles, Guillermo J

    2002-08-01

    We have used a novel method to conduct non-myeloablative stem cell transplantation (NST), making the following changes in previous methods: Use of the cheapest conditioning drugs, tailored number of apheresis sessions in the donors, elimination of ganciclovir and IgG, outpatient conduction when possible, diminished number of transfusions of blood products and diminished number of donor lymphocyte infusions. With this method, we have prospectively conducted 70 allografts in patients with different diseases: Chronic myelogenous leukemia, acute myelogenous leukemia, acute lymphoblastic leukemia, myelodysplasia, thalassemia major, relapsed Hodgkins disease, Blackfan-Diamond syndrome and aplastic anemia. In them, the median granulocyte recovery time to 0.5 x 10(9)/L was 11 d, whereas the median platelet recovery time to 20 x 10(9)/L was 12 d. Twenty patients did not need red blood cell transfusions and 17 did not need platelet transfusions. In 55 individuals (78%), the procedure could be completed fully on an outpatient basis. Follow-up times range between 30 and 800 d.: Four patients failed to engraft and recovered endogenous hemopoiesis; 16 patients (23%) developed acute graft versus-host disease (GVHD) whereas 28 (49%) developed chronic GVHD. Thirty two patients (47%) have died: 21 with a relapsing disease and seven as a result of GVHD; the median post-trasplant survival (SV) was 420 d., whereas the 12-mo. SV was 42%. The 100-day mortality was 3.8% and the transplant-related mortality was 14.2%. The median cost of the allografts was 18,000.00 US dollars. This method could be particularly adequate in developing countries, where very few individuals can afford the cost of a conventional bone marrow transplantation procedure.

  19. Conditions for diffusion-limited and reaction-limited recombination in nanostructured solar cells

    NASA Astrophysics Data System (ADS)

    Ansari-Rad, Mehdi; Anta, Juan A.; Arzi, Ezatollah

    2014-04-01

    The performance of Dye-sensitized solar cells (DSC) and related devices made of nanostructured semiconductors relies on a good charge separation, which in turn is achieved by favoring charge transport against recombination. Although both processes occur at very different time scales, hence ensuring good charge separation, in certain cases the kinetics of transport and recombination can be connected, either in a direct or an indirect way. In this work, the connection between electron transport and recombination in nanostructured solar cells is studied both theoretically and by Monte Carlo simulation. Calculations using the Multiple-Trapping model and a realistic trap distribution for nanostructured TiO2 show that for attempt-to-jump frequencies higher than 1011-1013 Hz, the system adopts a reaction limited (RL) regime, with a lifetime which is effectively independent from the speed of the electrons in the transport level. For frequencies lower than those, and depending on the concentration of recombination centers in the material, the system enters a diffusion-limited regime (DL), where the lifetime increases if the speed of free electrons decreases. In general, the conditions for RL or DL recombination depend critically on the time scale difference between recombination kinetics and free-electron transport. Hence, if the former is too rapid with respect to the latter, the system is in the DL regime and total thermalization of carriers is not possible. In the opposite situation, a RL regime arises. Numerical data available in the literature, and the behavior of the lifetime with respect to (1) density of recombination centers and (2) probability of recombination at a given center, suggest that a typical DSC in operation stays in the RL regime with complete thermalization, although a transition to the DL regime may occur for electrolytes or hole conductors where recombination is especially rapid or where there is a larger dispersion of energies of electron acceptors.

  20. Impact of KIR and HLA Genotypes on Outcomes after Reduced-Intensity Conditioning Hematopoietic Cell Transplantation

    PubMed Central

    Sobecks Ronald, M; Tao, Wang; Medhat, Askar; Gallagher Meighan, M; Michael, Haagenson; Stephen, Spellman; Marcelo, Fernandez-Vina; Karl-Johan, Malmberg; Carlheinz, Muller; Minoo, Battiwalla; James, Gajewski; Verneris Michael, R; Olle, Ringden; Marino Susana, R; Stella, Davies; Jason, Dehn; Martin, Bornhäuser; Yoshihiro, Inamoto; Ann, Woolfrey; Peter, Shaw; Marilyn, Pollack; Daniel, Weisdorf; Jeffrey, Miller; Hurley Carolyn, K; Lee Stephanie, J; Hsu Katharine, C

    2015-01-01

    Natural killer (NK) cells are regulated killer immunoglobulin-like receptor (KIR) interactions with HLA class I ligands. Several models of NK reactivity have been associated with improved outcomes following myeloablative allogeneic hematopoietic cell transplantation (HCT), but this issue has not been rigorously addressed in reduced-intensity conditioning (RIC) unrelated donor (URD) HCT. We studied 909 patients undergoing RIC-URD HCT. Patients with acute myeloid leukemia (AML, n=612) lacking ≥1 KIR ligands experienced higher grade III–IV acute graft-vs.-host disease (GvHD) (HR 1.6, 95%CI 1.16–2.28, p=0.005) compared to those with all ligands present. Absence of HLA-C2 for donor KIR2DL1 was associated with higher grade II–IV (HR 1.4, p=0.002) and III–IV acute GvHD (HR 1.5, p=0.01) compared to HLA-C2+patients. AML patients with KIR2DS1+, HLA-C2 homozygous donors had greater treatment-related mortality compared to others (HR 2.4, 95%CI 1.4–4.2, p=0.002), but did not experience lower relapse. There were no significant associations with outcomes for AML when assessing donor activating KIRs or centromeric KIR content, nor for any donor-recipient KIR-HLA assessments in patients with myelodysplastic syndrome (n=297). KIR-HLA combinations in RIC-URD HCT recapitulate some but not all KIR-HLA effects observed in myeloablative HCT. PMID:25960307

  1. Conditions for accurate Karl Fischer coulometry using diaphragm-free cells.

    PubMed

    Nordmark, U; Cedergren, A

    2000-01-01

    Factors influencing the extent of formation of oxidizable reduction products in coulometric cells used for Karl Fischer (KF) determination of water were investigated. For methanolic KF reagents buffered with imidazole (Im) or diethanolamine (DEA) (separately or in combination), three parameters were found to be of outmost importance: the cathodic current density, the pH, and the concentration of protonated base (ImH+ or DEAH+). For reagents buffered with only Im, the relative formation of oxidizable reduction products varied in the range 2-40%; i.e., 51-70 micrograms of water was found for a 50 micrograms water sample, depending on the above-mentioned parameters. The lowest values were observed for reagents having a pH around 10 in combination with cathodic current densities in the range 2000-5000 mA cm-2. For all the Imbuffered reagents investigated, the addition of modifiers such as chloroform, hexanol, and carbon tetrachloride was found to decrease the formation of oxidizable reduction products significantly. For example, a reagent buffered at pH 10 containing 1 M hexanol gave less than 0.3% formation in the current density interval from 200 to 4000 mA cm-2. The best reagents based on the above-mentioned modifiers were tested in the continuous coulometric mode with errors typically in the interval 0-0.5% using optimum conditions. One prerequisite for obtaining such small errors with diaphragm-free continuous coulometry is to use a cathode area no larger than 0.002 cm2. For some of the reagents based on both Im and DEA, the formation of oxidizable reduction products was close to zero at certain current densities, although the analytical performance was not as good as for the reagents buffered solely by Im due to longer conditioning and titration times.

  2. Transcript profiles in cortical cells of maize primary root during ethylene-induced lysigenous aerenchyma formation under aerobic conditions

    PubMed Central

    Takahashi, Hirokazu; Yamauchi, Takaki; Rajhi, Imene; Nishizawa, Naoko K.; Nakazono, Mikio

    2015-01-01

    Background and Aims Internal aeration is important for plants to survive during periods of waterlogging, and the ability to form aerenchyma contributes by creating a continuous gas space between the shoots and the roots. Roots of maize (Zea mays) react to prolonged waterlogging by forming aerenchyma in root cortical cells by programmed cell death (PCD) in response to ethylene. The aim of this study was to understand the molecular mechanisms of ethylene-induced aerenchyma formation by identifying genes that are either up- or downregulated by ethylene treatment in maize root cortical cells. Methods Three-day-old maize seedlings were treated with ethylene for several hours under aerobic conditions. Cortical cells were isolated from the primary roots using laser microdissection (LM), and transcript profiles with and without ethylene treatment were compared by microarray. In addition, the effect on ethylene-induced aerenchyma formation of diphenyleneiodonium (DPI), an inhibitor of NADPH oxidases, was examined in order to assess the involvement of reactive oxygen species (ROS). Key Results A total of 223 genes were identified whose transcript levels were significantly increased or decreased by ethylene treatment in root cortical cells under aerobic conditions. Subsequent tissue-specific quantitative reverse-transcription PCR analyses revealed that ethylene increased the transcript levels of genes related to ethylene signalling in all of the root tissues examined (stelar cells, cortical cells and outer cell layers), whereas it increased the transcript levels of genes related to cell wall modification and proteolysis specifically in the cortical cells. DPI treatment inhibited the ethylene-induced aerenchyma formation and suppressed expression of some cell wall modification-related genes. Conclusions Several genes related to cell wall modification and proteolysis are specifically up- or downregulated in cortical cells during lysigenous aerenchyma formation under aerobic

  3. Identification of a New Cell Population Constitutively Circulating in Healthy Conditions and Endowed with a Homing Ability Toward Injured Sites

    PubMed Central

    Lo Sicco, Claudia; Tasso, Roberta; Reverberi, Daniele; Cilli, Michele; Pfeffer, Ulrich; Cancedda, Ranieri

    2015-01-01

    Stem and progenitor cells are the critical units for tissue maintenance, regeneration, and repair. The activation of regenerative events in response to tissue injury has been correlated with mobilization of tissue-resident progenitor cells, which is functional to the wound healing process. However, until now there has been no evidence for the presence of cells with a healing capacity circulating in healthy conditions. We identified a rare cell population present in the peripheral blood of healthy mice that actively participates in tissue repair. These Circulating cells, with a Homing ability and involved in the Healing process (CH cells), were identified by an innovative flowcytometry strategy as small cells not expressing CD45 and lineage markers. Their transcriptome profile revealed that CH cells are unique and present a high expression of key pluripotency- and epiblast-associated genes. More importantly, CH-labeled cells derived from healthy Red Fluorescent Protein (RFP)-transgenic mice and systemically injected into syngeneic fractured wild-type mice migrated and engrafted in wounded tissues, ultimately differentiating into tissue-specific cells. Accordingly, the number of CH cells in the peripheral blood rapidly decreased following femoral fracture. These findings uncover the existence of constitutively circulating cells that may represent novel, accessible, and versatile effectors of therapeutic tissue regeneration. PMID:26560420

  4. The Autophagoproteasome a Novel Cell Clearing Organelle in Baseline and Stimulated Conditions

    PubMed Central

    Lenzi, Paola; Lazzeri, Gloria; Biagioni, Francesca; Busceti, Carla L.; Gambardella, Stefano; Salvetti, Alessandra; Fornai, Francesco

    2016-01-01

    Protein clearing pathways named autophagy (ATG) and ubiquitin proteasome (UP) control homeostasis within eukaryotic cells, while their dysfunction produces neurodegeneration. These pathways are viewed as distinct biochemical cascades occurring within specific cytosolic compartments owing pathway-specific enzymatic activity. Recent data strongly challenged the concept of two morphologically distinct and functionally segregated compartments. In fact, preliminary evidence suggests the convergence of these pathways to form a novel organelle named autophagoproteasome. This is characterized in the present study by using a cell line where, mTOR activity is upregulated and autophagy is suppressed. This was reversed dose-dependently by administering the mTOR inhibitor rapamycin. Thus, we could study autophagoproteasomes when autophagy was either suppressed or stimulated. The occurrence of autophagoproteasome was shown also in non-human cell lines. Ultrastructural morphometry, based on the stochiometric binding of immunogold particles allowed the quantitative evaluation of ATG and UP component within autophagoproteasomes. The number of autophagoproteasomes increases following mTOR inhibition. Similarly, mTOR inhibition produces overexpression of both LC3 and P20S particles. This is confirmed by the fact that the ratio of free vs. autophagosome-bound LC3 is similar to that measured for P20S, both in baseline conditions and following mTOR inhibition. Remarkably, within autophagoproteasomes there is a slight prevalence of ATG compared with UP components for low rapamycin doses, whereas for higher rapamycin doses UP increases more than ATG. While LC3 is widely present within cytosol, UP is strongly polarized within autophagoproteasomes. These fine details were evident at electron microscopy but could not be deciphered by using confocal microscopy. Despite its morphological novelty autophagoproteasomes appear in the natural site where clearing pathways (once believed to be

  5. The Autophagoproteasome a Novel Cell Clearing Organelle in Baseline and Stimulated Conditions.

    PubMed

    Lenzi, Paola; Lazzeri, Gloria; Biagioni, Francesca; Busceti, Carla L; Gambardella, Stefano; Salvetti, Alessandra; Fornai, Francesco

    2016-01-01

    Protein clearing pathways named autophagy (ATG) and ubiquitin proteasome (UP) control homeostasis within eukaryotic cells, while their dysfunction produces neurodegeneration. These pathways are viewed as distinct biochemical cascades occurring within specific cytosolic compartments owing pathway-specific enzymatic activity. Recent data strongly challenged the concept of two morphologically distinct and functionally segregated compartments. In fact, preliminary evidence suggests the convergence of these pathways to form a novel organelle named autophagoproteasome. This is characterized in the present study by using a cell line where, mTOR activity is upregulated and autophagy is suppressed. This was reversed dose-dependently by administering the mTOR inhibitor rapamycin. Thus, we could study autophagoproteasomes when autophagy was either suppressed or stimulated. The occurrence of autophagoproteasome was shown also in non-human cell lines. Ultrastructural morphometry, based on the stochiometric binding of immunogold particles allowed the quantitative evaluation of ATG and UP component within autophagoproteasomes. The number of autophagoproteasomes increases following mTOR inhibition. Similarly, mTOR inhibition produces overexpression of both LC3 and P20S particles. This is confirmed by the fact that the ratio of free vs. autophagosome-bound LC3 is similar to that measured for P20S, both in baseline conditions and following mTOR inhibition. Remarkably, within autophagoproteasomes there is a slight prevalence of ATG compared with UP components for low rapamycin doses, whereas for higher rapamycin doses UP increases more than ATG. While LC3 is widely present within cytosol, UP is strongly polarized within autophagoproteasomes. These fine details were evident at electron microscopy but could not be deciphered by using confocal microscopy. Despite its morphological novelty autophagoproteasomes appear in the natural site where clearing pathways (once believed to be

  6. Primary Human Uterine Leiomyoma Cell Culture Quality Control: Some Properties of Myometrial Cells Cultured under Serum Deprivation Conditions in the Presence of Ovarian Steroids

    PubMed Central

    Sumikawa, Joana Tomomi; Batista, Fabrício Pereira; Paredes-Gamero, Edgar J.; Girão, Manoel J. B. C.; Oliva, Maria Luiza V.

    2016-01-01

    Cell culture is considered the standard media used in research to emulate the in vivo cell environment. Crucial in vivo experiments cannot be conducted in humans and depend on in vitro methodologies such as cell culture systems. However, some procedures involving the quality control of cells in culture have been gradually neglected by failing to acknowledge that primary cells and cell lines change over time in culture. Thus, we report methods based on our experience for monitoring primary cell culture of human myometrial cells derived from uterine leiomyoma. We standardized the best procedure of tissue dissociation required for the study of multiple genetic marker systems that include species-specific antigens, expression of myofibroblast or myoblast markers, growth curve, serum deprivation, starvation by cell cycle synchronization, culture on collagen coated plates, and 17 β-estradiol (E2) and progesterone (P4) effects. The results showed that primary myometrial cells from patients with uterine leiomyoma displayed myoblast phenotypes before and after in vitro cultivation, and leiomyoma cells differentiated into mature myocyte cells under the appropriate differentiation-inducing conditions (serum deprivation). These cells grew well on collagen coated plates and responded to E2 and P4, which may drive myometrial and leiomyoma cells to proliferate and adhere into a focal adhesion complex involvement in a paracrine manner. The establishment of these techniques as routine procedures will improve the understanding of the myometrial physiology and pathogenesis of myometrium-derived diseases such as leiomyoma. Mimicking the in vivo environment of fibrotic conditions can prevent false results and enhance results that are based on cell culture integrity. PMID:27391384

  7. Expression pattern of neurotrophins and their receptors during neuronal differentiation of adipose-derived stem cells in simulated microgravity condition

    PubMed Central

    Zarrinpour, Vajiheh; Hajebrahimi, Zahra; Jafarinia, Mojtaba

    2017-01-01

    Objective(s): Studies have confirmed that microgravity, as a mechanical factor, influences both differentiation and function of mesenchymal stem cells. Here we investigated the effects of simulated microgravity on neural differentiation of human adipose-derived stem cells (ADSCs). Materials and Methods: We have used a fast rotating clinostat (clinorotation) to simulate microgravity condition. Real-time PCR and flow cytometry analysis were used to evaluate the regulation of neurotrophins, their receptors, and neural markers by simulated microgravity and their impact on neural differentiation of cells. Results: Our data revealed that simulation microgravity up-regulated the expression of MAP-2, BDNF, TrkB, NT-3, and TrkC both before and after neural differentiation. Also, the neural cells derived from ADSCs in microgravity condition expressed more MAP-2, GFAP, and synaptophysin protein in comparison to the 1G control. Conclusion: We showed that simulated microgravity can enhance the differentiation of mesenchymal stem cells into neurons. Our findings provide a new strategy for differentiation of ADSCs to neural-like cells and probably other cell lineages. Meanwhile, microgravity simulation had no adverse effects on the viability of the cells and could be used as a new environment to successfully manipulate cells. PMID:28293395

  8. Proton exchange membrane fuel cell degradation under close to open-circuit conditions. Part I: In situ diagnosis

    NASA Astrophysics Data System (ADS)

    Wu, Jinfeng; Yuan, Xiao-Zi; Martin, Jonathan J.; Wang, Haijiang; Yang, Daijun; Qiao, Jinli; Ma, Jianxin

    Durability of polymer exchange membrane (PEM) fuel cells under a wide range of operational conditions has been generally identified as one of the top technical gaps that need to be overcome for the acceptance of this fuel cell technology as a commercially viable power source, especially for automotive and portable applications. In this study, a 1200 h lifetime test was conducted with a six-cell PEM fuel cell stack under close to open-circuit conditions. In situ measurements of the hydrogen crossover rate through the membrane, high frequency resistance and electrochemically active surface area of each single cell, in combination with cell polarization curves, were used to investigate the degradation mechanisms. Direct gas mass spectrometry of the cathode exhaust gas indicated the formation of HF, H 2O 2 and CO 2 during the durability testing. The overall cell degradation rate under this accelerated stress testing is approximately 0.128 mV h -1. The cell degradation rate for the first 800 h is much lower than that after 800 h, which may result from the dominance of different degradation mechanisms. For the first period, the degradation of fuel cell performance was mainly attributed to catalyst decay, while the subsequent dramatic degradation is likely caused by membrane failure.

  9. In vitro cementoblast-like differentiation of postmigratory neural crest-derived p75{sup +} stem cells with dental follicle cell conditioned medium

    SciTech Connect

    Wen, Xiujie; Liu, Luchuan; Deng, Manjing; Liu, Rui; Zhang, Li; Nie, Xin

    2015-09-10

    Cranial neural crest-derived cells (CNCCs) play important role in epithelial–mesenchymal interactions during tooth morphogenesis. However, the heterogeneity of CNCCs and their tendency to spontaneously differentiate along smooth muscle or osteoblast lineages in vitro limit further understanding of their biological properties. We studied the differentiation properties of isolated rat embryonic postmigratory CNCCs, expressing p75 neurotrophin receptor (p75NTR). These p75NTR positive (p75{sup +}) CNCCs, isolated using fluorescence activated cell sorter, exhibited fibroblast-like morphology and characteristics of mesenchymal stem cells. Incubation of p75{sup +} CNCCs in dental follicle cell conditioned medium (DFCCM) combined with dentin non-collagenous proteins (dNCPs), altered their morphological features to cementoblast-like appearance. These cells also showed low proliferative activity, high ALP activity and significantly increased calcified nodule formation. Markers related to mineralization or specific to cementoblast lineage were highly expressed in dNCPs/DFCCM-treated p75{sup +} cells, suggesting their differentiation along cementoblast-like lineage. p75{sup +} stem cells selected from postmigratory CNCCs represent a pure stem cell population and could be used as a stem cell model for in vitro studies due to their intrinsic ability to differentiate to neuronal cells and transform from neuroectoderm to ectomesenchyme. They can provide a potential stem cell resource for tooth engineering studies and help to further investigate mechanisms of epithelial–mesenchymal interactions in tooth morphogenesis. - Highlights: • Cranial neural crest-derived cells (CNCCs) take part in tooth morphogenesis. • positive (p75{sup +}) CNCCs are fibroblast-like and resemble mesenchymal stem cells. • p75{sup +} CNCCs in dental follicle cell medium (DFCCM/dNCP) appear like cementoblasts. • DFCCM/dNCP-treated p75{sup +} cells express cementoblast specific mineralization

  10. B Cells Are Critical to T-cell-Mediated Antitumor Immunity Induced by a Combined Immune-Stimulatory/Conditionally Cytotoxic Therapy for Glioblastoma12

    PubMed Central

    Candolfi, Marianela; Curtin, James F; Yagiz, Kader; Assi, Hikmat; Wibowo, Mia K; Alzadeh, Gabrielle E; Foulad, David; Muhammad, AKM G; Salehi, Sofia; Keech, Naomi; Puntel, Mariana; Liu, Chunyan; Sanderson, Nicholas R; Kroeger, Kurt M; Dunn, Robert; Martins, Gislaine; Lowenstein, Pedro R; Castro, Maria G

    2011-01-01

    We have demonstrated that modifying the tumor microenvironment through intratumoral administration of adenoviral vectors (Ad) encoding the conditional cytotoxic molecule, i.e., HSV1-TK and the immune-stimulatory cytokine, i.e., fms-like tyrosine kinase 3 ligand (Flt3L) leads to T-cell-dependent tumor regression in rodent models of glioblastoma. We investigated the role of B cells during immune-mediated glioblastoma multiforme regression. Although treatment with Ad-TK+Ad-Flt3L induced tumor regression in 60% of wild-type (WT) mice, it completely failed in B-cell-deficient Igh6-/- mice. Tumor-specific T-cell precursors were detected in Ad-TK+Ad-Flt3L-treated WT mice but not in Igh6-/- mice. The treatment also failed in WT mice depleted of total B cells or marginal zone B cells. Because we could not detect circulating antibodies against tumor cells and the treatment was equally efficient in WT mice and in mice with B-cell-specific deletion of Prdm 1 (encoding Blimp-1), in which B cells are present but unable to fully differentiate into antibody-secreting plasma cells, tumor regression in this model is not dependent on B cells' production of tumor antigen-specific immunoglobulins. Instead, B cells seem to play a role as antigen-presenting cells (APCs). Treatment with Ad-TK+Ad-Flt3L led to an increase in the number of B cells in the cervical lymph nodes, which stimulated the proliferation of syngeneic T cells and induced clonal expansion of antitumor T cells. Our data show that B cells act as APCs, playing a critical role in clonal expansion of tumor antigen-specific T cells and brain tumor regression. PMID:22028620

  11. Neuro-Immune Interactions for Cell-based Model under the Conditions of Irradiation

    NASA Astrophysics Data System (ADS)

    Zhu, Huiyang; Deng, Yulin; Lei, Runhong; Ma, Hong; Zhuang, Fengyuan

    AIM: To explore the proliferation effect of the SH-SY5Y cells which is damaged by the radiation on the Jurkat cells and THP-1 cells. METHODS: The first, SH-SY5Y cells was on the dose of 7 Gy γ-ray radiation, then the cells were cultured at 37℃ until 24 hours later, and the culture medium was changed. The second, culture of SH-SY5Y cells was removed, and it was used for Jurkat cells and THP-1 cells. The third, Jurkat cells and THP-1 cells were cultured until 24 hours later. At this moment the cell number was counted by the MTS. RESULTS: On the MTS results, the number of Jurkat cells in the experimental group was significantly lower than the control group, but the number of THP-1 cells in the experimental group was much higher than the control group. CONCLUSION: The 24 hours medium culture from SH-SY5Y cells which was damaged by radiation would Inhibition of the Jurkat cell growth, but it would Promote the growth of the THP-1 cell.

  12. Three-dimensional spherical spatial boundary conditions differentially regulate osteogenic differentiation of mesenchymal stromal cells

    PubMed Central

    Lo, Yin-Ping; Liu, Yi-Shiuan; Rimando, Marilyn G.; Ho, Jennifer Hui-Chun; Lin, Keng-hui; Lee, Oscar K.

    2016-01-01

    The spatial boundary condition (SBC) arising from the surrounding microenvironment imposes specific geometry and spatial constraints that affect organogenesis and tissue homeostasis. Mesenchymal stromal cells (MSCs) sensitively respond to alterations of mechanical cues generated from the SBC. However, mechanical cues provided by a three-dimensional (3D) environment are deprived in a reductionist 2D culture system. This study investigates how SBC affects osteogenic differentiation of MSCs using 3D scaffolds with monodispersed pores and homogenous spherical geometries. MSCs cultured under SBCs with diameters of 100 and 150 μm possessed the greatest capability of osteogenic differentiation. This phenomenon was strongly correlated with MSC morphology, organization of actin cytoskeleton, and distribution of focal adhesion involving α2 and α5 integrins. Further silencing either α2 or α5 integrin significantly reduced the above mentioned mechanosensitivity, indicating that the α2 and α5 integrins as mechano-sensitive molecules mediate MSCs’ ability to provide enhanced osteogenic differentiation in response to different spherical SBCs. Taken together, the findings provide new insights regarding how MSCs respond to mechanical cues from the surrounding microenvironment in a spherical SBC, and such biophysical stimuli should be taken into consideration in tissue engineering and regenerative medicine in conjunction with biochemical cues. PMID:26884253

  13. Conditioned medium derived from umbilical cord mesenchymal stem cells regenerates atrophied muscles.

    PubMed

    Kim, Mi Jin; Kim, Z-Hun; Kim, Sun-Mi; Choi, Yong-Soo

    2016-10-01

    We investigated the regenerative effects and regulatory mechanisms of human umbilical cord mesenchymal stem cells (UC-MSCs)-derived conditioned medium (CM) in atrophied muscles using an in vivo model. To determine the appropriate harvest point of UC-CM, active factor content was analyzed in the secretome over time. A muscle atrophy model was induced in rats by hindlimb suspension (HS) for 2 weeks. Next, UC-CM was injected directly into the soleus muscle of both hind legs to assess its regenerative efficacy on atrophy-related factors after 1 week of HS. During HS, muscle mass and muscle fiber size were significantly reduced by over 2-fold relative to untreated controls. Lactate accumulation within the muscles was similarly increased. By contrast, all of the above analytical factors were significantly improved in HS-induced rats by UC-CM injection compared with saline injection. Furthermore, the expression levels of desmin and skeletal muscle actin were significantly elevated by UC-CM treatment. Importantly, UC-CM effectively suppressed expression of the atrophy-related ubiquitin E3-ligases, muscle ring finger 1 and muscle atrophy F-box by 2.3- and 2.1-fold, respectively. UC-CM exerted its actions by stimulating the phosphoinositol-3-kinase (PI3K)/Akt signaling cascade. These findings suggest that UC-CM provides an effective stimulus to recover muscle status and function in atrophied muscles.

  14. Performance of the Fuel Conditioning Facility electronic in-cell mass balances

    SciTech Connect

    Orechwa, Y.; Bucher, R.G.

    1996-05-01

    An approach to error estimation and measurement control in the analysis of the balance measurements of mass standards on the in-cell electronic mass balances of the Fuel Conditioning Facility is presented. In light of measurement data from one year of operation, the algorithms proposed are evaluated. The need to take into account the effects of facility operations on the estimates of measurement uncertainty is demonstrated. In the case of a newly installed balance, where no historical data exists, an ad hoc procedure of adding a term which takes into account the operational variability is proposed. This procedure allows a sufficiently long operation so as to collect data for the estimate of the contribution of operational effects to the uncertainty estimate. An algorithm for systematically taking into account historical data is developed and demonstrated for two balances over two calibration periods. The algorithm, both asymptotically and in the two samples cases, has the necessary desirable properties for estimating the uncertainty in the measurements of the balances.

  15. Increased power generation from primary sludge by a submersible microbial fuel cell and optimum operational conditions.

    PubMed

    Vologni, Valentina; Kakarla, Ramesh; Angelidaki, Irini; Min, Booki

    2013-05-01

    Microbial fuel cells (MFCs) have received attention as a promising renewable energy technology for waste treatment and energy recovery. We tested a submersible MFC with an innovative design capable of generating a stable voltage of 0.250 ± 0.008 V (with a fixed 470 Ω resistor) directly from primary sludge. In a polarization test, the maximum power density was 0.18 W/m(2) at a current density of 0.8 A/m(2) with an external resistor of 300 Ω. The anodic solution of the primary sludge needs to be adjusted to a pH 7 for high power generation. The modified primary sludge with an added phosphate buffer prolonged the current generation and increased the power density by 7 and 1.5 times, respectively, in comparison with raw primary sludge. These findings suggest that energy recovery from primary sludge can be maximized using an advanced MFC system with optimum conditions.

  16. Inhibition of Murine Pulmonary Microvascular Endothelial Cell Apoptosis Promotes Recovery of Barrier Function under Septic Conditions

    PubMed Central

    Wang, Lefeng; Mehta, Sanjay; Brock, Michael

    2017-01-01

    Sepsis is characterized by injury of the pulmonary microvasculature and the pulmonary microvascular endothelial cells (PMVEC), leading to barrier dysfunction and acute respiratory distress syndrome (ARDS). Our recent work identified a strong correlation between PMVEC apoptosis and microvascular leak in septic mice in vivo, but the specific role of apoptosis in septic PMVEC barrier dysfunction remains unclear. Thus, we hypothesize that PMVEC apoptosis is likely required for PMVEC barrier dysfunction under septic conditions in vitro. Septic stimulation (mixture of tumour necrosis factor α, interleukin 1β, and interferon γ [cytomix]) of isolated murine PMVEC resulted in a significant loss of barrier function as early as 4 h after stimulation, which persisted until 24 h. PMVEC apoptosis, as reflected by caspase activation, DNA fragmentation, and loss of membrane polarity, was first apparent at 8 h after cytomix. Pretreatment of PMVEC with the pan-caspase inhibitor Q-VD significantly decreased septic PMVEC apoptosis and was associated with reestablishment of PMVEC barrier function at 16 and 24 h after stimulation but had no effect on septic PMVEC barrier dysfunction over the first 8 h. Collectively, our data suggest that early septic murine PMVEC barrier dysfunction driven by proinflammatory cytokines is not mediated through apoptosis, but PMVEC apoptosis contributes to late septic PMVEC barrier dysfunction. PMID:28250575

  17. Increased efficiency of mammalian somatic cell hybrid production under microgravity conditions during ballistic rocket flight

    NASA Technical Reports Server (NTRS)

    Schnettler, R.; Gessner, P.; Zimmermann, U.; Neil, G. A.; Urnovitz, H. B.

    1989-01-01

    The electrofusion of hybridoma cell lines under short-duration microgravity during a flight of the TEXUS 18 Black Brand ballistic sounding rocket at Kiruna, Sweden is reported. The fusion partners, growth medium, cell fusion medium, cell fusion, cell viability in the fusion medium, and postfusion cell culture are described, and the rocket, cell fusion chamber, apparatus, and module are examined. The experimental timeline, the effects of fusion medium and incubation time on cell viability and hybrid yields, and the effect of microgravity on hybrid yields are considered.

  18. Conditions for the generation of cytotoxic CD4+ Th cells that enhance CD8+ CTL-mediated tumor regression

    PubMed Central

    Li, Kunyu; Baird, Margaret; Yang, Jianping; Jackson, Chris; Ronchese, Franca; Young, Sarah

    2016-01-01

    Adoptive cell therapies (ACTs) using tumor-reactive T cells have shown clinical benefit and potential for cancer treatment. While the majority of the current ACT are focused on using CD8+ cytotoxic T lymphocytes (CTL), others have shown that the presence of tumor-reactive CD4+ T helper (Th) cells can greatly enhance the anti-tumor activity of CD8+ CTL. However, difficulties in obtaining adequate numbers of CD4+ Th cells through in vitro expansion can limit the application of CD4 Th cells in ACT. This study aims to optimize the culture conditions for mouse CD4 T cells to provide basic information for animal studies of ACT using CD4 T cells. Taking advantage of the antigen-specificity of CD4+ Th cells from OT-II transgenic mice, we examined different methodologies for generating antigen-specific CD4+ Th1 cells in vitro. We found that cells grown in complete advanced-DMEM/F12 medium supplemented with low-dose IL-2 and IL-7 induced substantial cell expansion. These Th cells were Th1-like, as they expressed multiple Th1-cytokines and exhibited antigen-specific cytotoxicity. In addition co-transfer of these CD4+ Th1-like cells with CD8+ CTL significantly enhanced tumor regression, leading to complete cure in 80% of mice bearing established B16-OVA. These observations indicate that the CD4+ Th1-like cells generated using the method we optimized are functionally active to eliminate their target cells, and can also assist CD8+ CTL to enhance tumor regression. The findings of this study provide valuable data for further research into in vitro expansion of CD4+ Th1-like cells, with potential applications to cancer treatment involving ACT. PMID:27588200

  19. Cell Therapy Augments Functional Recovery Subsequent to Spinal Cord Injury under Experimental Conditions

    PubMed Central

    Sabapathy, Vikram; Tharion, George; Kumar, Sanjay

    2015-01-01

    The spinal cord injury leads to enervation of normal tissue homeostasis ultimately leading to paralysis. Until now there is no proper cure for the treatment of spinal cord injury. Recently, cell therapy in animal spinal cord injury models has shown some progress of recovery. At present, clinical trials are under progress to evaluate the efficacy of cell transplantation for the treatment of spinal cord injury. Different types of cells such as pluripotent stem cells derived neural cells, mesenchymal stromal cells, neural stem cells, glial cells are being tested in various spinal cord injury models. In this review we highlight both the advances and lacuna in the field of spinal cord injury by discussing epidemiology, pathophysiology, molecular mechanism, and various cell therapy strategies employed in preclinical and clinical injury models and finally we discuss the limitations and ethical issues involved in cell therapy approach for treating spinal cord injury. PMID:26240569

  20. Arg tyrosine kinase modulates TGF-β1 production in human renal tubular cells under high-glucose conditions.

    PubMed

    Torsello, Barbara; Bianchi, Cristina; Meregalli, Chiara; Di Stefano, Vitalba; Invernizzi, Lara; De Marco, Sofia; Bovo, Giorgio; Brivio, Rinaldo; Strada, Guido; Bombelli, Silvia; Perego, Roberto A

    2016-08-01

    Renal tubular cells are involved in the tubular interstitial fibrosis observed in diabetic nephropathy. It is debated whether epithelial-mesenchymal transition (EMT) affects tubular cells, which under high-glucose conditions overproduce transforming growth factor-β (TGF-β), a fibrogenic cytokine involved in interstitial fibrosis development. Our study investigated the involvement of non-receptor tyrosine kinase Arg (also called Abl2) in TGF-β production. Human primary tubular cell cultures exposed to high-glucose conditions were used. These cells showed an elongated morphology, stress fibers and vimentin increment but maintained most of the epithelial marker expression and distribution. In these cells exposed to high glucose, which overexpressed and secreted active TGF-β1, Arg protein and activity was downregulated. A further TGF-β1 increase was induced by Arg silencing with siRNA, as with the Arg tyrosine kinase inhibitor Imatinib. In the cells exposed to high glucose, reactive oxygen species (ROS)-dependent Arg kinase downregulation induced both RhoA activation, through p190RhoGAPA (also known as ARHGAP35) modulation, and proteasome activity inhibition. These data evidence a new specific involvement of Arg kinase into the regulation of TGF-β1 expression in tubular cells under high-glucose conditions and provide cues for new translational approaches in diabetic nephropathy.

  1. (211)Astatine-Conjugated Monoclonal CD45 Antibody-Based Nonmyeloablative Conditioning for Stem Cell Gene Therapy.

    PubMed

    Burtner, Christopher R; Chandrasekaran, Devikha; Santos, Erlinda B; Beard, Brian C; Adair, Jennifer E; Hamlin, Donald K; Wilbur, D Scott; Sandmaier, Brenda M; Kiem, Hans-Peter

    2015-06-01

    Most hematopoietic stem cell gene therapy studies require host conditioning to allow for efficient engraftment of gene-modified cells. Conditioning regimens with lower treatment-related toxicities are especially relevant for the treatment of nonmalignant blood disorders, such as hemoglobinopathies and immunodeficiencies, and for patients who are otherwise ineligible for conventional high-dose conditioning. Radioimmunotherapy, which employs an α- or a β-emitting radionuclide conjugated to a targeting antibody, is effective for delivering cytotoxic doses of radiation to a cell type of interest while minimizing off-target toxicity. Here, we demonstrate the feasibility of using a nonmyeloablative dose of a monoclonal anti-CD45 antibody conjugated to the α-emitter Astatine-211 ((211)At) to promote engraftment of an autologous gene-modified stem cell graft in the canine model. The doses used provided myelosuppression with rapid autologous recovery and minimal off-target toxicity. Engraftment levels were low in all dogs and reflected the low numbers of gene-modified cells infused. Our data suggest that a cell dose exceeding 1×10(6) cells/kg be used with nonmyeloablative doses of (211)At-anti-CD45 monoclonal antibodies for sustained engraftment in the dog model.

  2. Patient-specific modeling and analysis of dynamic behavior of individual sickle red blood cells under hypoxic conditions

    NASA Astrophysics Data System (ADS)

    Li, Xuejin; Du, E.; Li, Zhen; Tang, Yu-Hang; Lu, Lu; Dao, Ming; Karniadakis, George

    2015-11-01

    Sickle cell anemia is an inherited blood disorder exhibiting heterogeneous morphology and abnormal dynamics under hypoxic conditions. We developed a time-dependent cell model that is able to simulate the dynamic processes of repeated sickling and unsickling of red blood cells (RBCs) under physiological conditions. By using the kinetic cell model with parameters derived from patient-specific data, we present a mesoscopic computational study of the dynamic behavior of individual sickle RBCs flowing in a microfluidic channel with multiple microgates. We investigate how individual sickle RBCs behave differently from healthy ones in channel flow, and analyze the alteration of cellular behavior and response to single-cell capillary obstruction induced by cell rheologic rigidification and morphological change due to cell sickling under hypoxic conditions. We also simulate the flow dynamics of sickle RBCs treated with hydroxyurea (HU) and quantify the relative enhancement of hemodynamic performance of HU. This work was supported by the National Institutes of Health (NIH) Grant U01HL114476.

  3. Thyroid Cells Exposed to Simulated Microgravity Conditions - Comparison of the Fast Rotating Clinostat and the Random Positioning Machine

    NASA Astrophysics Data System (ADS)

    Warnke, Elisabeth; Kopp, Sascha; Wehland, Markus; Hemmersbach, Ruth; Bauer, Johann; Pietsch, Jessica; Infanger, Manfred; Grimm, Daniela

    2016-06-01

    The ground-based facilities 2D clinostat (CN) and Random Positioning Machine (RPM) were designed to simulate microgravity conditions on Earth. With support of the CORA-ESA-GBF program we could use both facilities to investigate the impact of simulated microgravity on normal and malignant thyroid cells. In this review we report about the current knowledge of thyroid cancer cells and normal thyrocytes grown under altered gravity conditions with a special focus on growth behaviour, changes in the gene expression pattern and protein content, as well as on altered secretion behaviour of the cells. We reviewed data obtained from normal thyrocytes and cell lines (two poorly differentiated follicular thyroid cancer cell lines FTC-133 and ML-1, as well as the normal thyroid cell lines Nthy-ori 3-1 and HTU-5). Thyroid cells cultured under conditions of simulated microgravity (RPM and CN) and in Space showed similar changes with respect to spheroid formation. In static 1 g control cultures no spheroids were detectable. Changes in the regulation of cytokines are discussed to be involved in MCS (multicellular spheroids) formation. The ESA-GBF program helps the scientists to prepare future spaceflight experiments and furthermore, it might help to identify targets for drug therapy against thyroid cancer.

  4. 211Astatine-Conjugated Monoclonal CD45 Antibody-Based Nonmyeloablative Conditioning for Stem Cell Gene Therapy

    PubMed Central

    Burtner, Christopher R.; Chandrasekaran, Devikha; Santos, Erlinda B.; Beard, Brian C.; Adair, Jennifer E.; Hamlin, Donald K.; Wilbur, D. Scott; Sandmaier, Brenda M.

    2015-01-01

    Abstract Most hematopoietic stem cell gene therapy studies require host conditioning to allow for efficient engraftment of gene-modified cells. Conditioning regimens with lower treatment-related toxicities are especially relevant for the treatment of nonmalignant blood disorders, such as hemoglobinopathies and immunodeficiencies, and for patients who are otherwise ineligible for conventional high-dose conditioning. Radioimmunotherapy, which employs an α- or a β-emitting radionuclide conjugated to a targeting antibody, is effective for delivering cytotoxic doses of radiation to a cell type of interest while minimizing off-target toxicity. Here, we demonstrate the feasibility of using a nonmyeloablative dose of a monoclonal anti-CD45 antibody conjugated to the α-emitter Astatine-211 (211At) to promote engraftment of an autologous gene-modified stem cell graft in the canine model. The doses used provided myelosuppression with rapid autologous recovery and minimal off-target toxicity. Engraftment levels were low in all dogs and reflected the low numbers of gene-modified cells infused. Our data suggest that a cell dose exceeding 1×106 cells/kg be used with nonmyeloablative doses of 211At-anti-CD45 monoclonal antibodies for sustained engraftment in the dog model. PMID:25919226

  5. In vivo imaging studies of the effect of recipient conditioning, donor cell phenotype and antigen disparity on homing of haematopoietic cells to the bone marrow.

    PubMed

    Askenasy, Nadir; Farkas, Daniel L

    2003-02-01

    Homing of transplanted bone marrow cells (BMC) to the host bone marrow (BM) is the first step of engraftment towards durable multilineage haematopoietic reconstitution. We used an in vivo assay to track PKH-labelled cells in the BM of mice after transplantation, using fluorescence microscopy through an optical window placed over the distal femoral epiphysis. Within hours after intravenous injection, the cells moved in and out the femur, and were mobile within the marrow space. One hour after injection of whole BMC into non-conditioned syngeneic and allogeneic recipients, the homing efficiencies (HE) were 1.23 +/- 0.14% and 0.12 +/- 0.02% respectively (P < 0.001). Irrespective of antigen disparity, the number of PKH-labelled cells in the femur decreased by 30% and 50% after 1 and 3 d respectively (P < 0.001). Similar HE of naïve and irradiated cells suggested that the majority of cells (> 80%) were quiescent in the BM during the first 3 d. HE were twofold higher in busulphan-myeloablated recipients (P < 0.001 vs non-conditioned), and allogeneic transplantation resulted in 84 +/- 9% donor chimaerism at 4 weeks. The HE of lin- cells was 16-fold higher than that of lin+ cells (P < 0.001), and the subset of lin- SCA-1+ cells was 4.6-fold higher in the BM-homed cell population (P < 0.001 vs lin- cells). Approximately 1,500 of the BM-homed cells rescued 62-71% secondary syngeneic and allogeneic myeloablated recipients. Strikingly, the HE could be predicted during the first 3 d after transplantation by correcting the measurements performed in vivo for the enrichment of progenitors in donor inoculum, donor-recipient antigen disparity and myeloablative conditioning.

  6. Differential Sulfation Remodelling of Heparan Sulfate by Extracellular 6-O-sulfatases Regulates Fibroblast Growth Factor-induced Boundary Formation By Glial Cells: Implications for Glial Cell Transplantation

    PubMed Central

    Higginson, Jennifer R; Thompson, Sophie M; Santos-Silva, Alessandra; Guimond, Scott E; Turnbull, Jeremy E; Barnett, Susan C

    2012-01-01

    Previously, it has been shown that rat Schwann cells (SCs), but not olfactory ensheathing cells (OECs), form a boundary with astrocytes, due to a SC-specific secreted factor. Here, we identify highly sulfated heparan sulfates (HS), and fibroblast growth factors (FGF) 1 and FGF9, as possible determinants of boundary formation induced by rat SCs. Disaccharide analysis of HS in SC and rat OEC conditioned medium showed that SCs secrete more highly sulfated HS than OECs. The dependence of the boundary-forming activity on high levels of sulfation was confirmed using a panel of semi-synthetic modified heparins with variable levels of sulfation. Furthermore, extracellular HS 6-O-endosulfatase enzymes, Sulf 1 and Sulf 2, were expressed at a significantly lower level by SCs compared to OECs and siRNA reduction of Sulfs in OECs was, in itself, sufficient to induce boundary formation. This demonstrates a key role for remodelling (reduction) of HS 6-O-sulfation by OECs to suppress boundary formation, in comparison to SCs. Furthermore, specific anti-FGF1 and FGF9 antibodies disrupted SC/astrocyte boundary formation, supporting a role for an HS sulfation-dependent FGF signalling mechanism via FGF receptors (FGFR) on astrocytes. We propose a model in which FGF1 and FGF9 signalling is differentially modulated by patterns of glial cell HS sulfation, dependent on Sulf 1 and Sulf 2 expression, to control FGFR3-IIIb mediated astrocytic responses. Moreover, these data suggest manipulation of HS sulfation after CNS injury as a potential novel approach for therapeutic intervention in CNS repair. PMID:23136428

  7. Generation of a conditionally immortalized myeloid progenitor cell line requiring the presence of both interleukin-3 and stem cell factor to survive and proliferate.

    PubMed

    Lee, Candy; Evans, Caroline A; Spooncer, Elaine; Pierce, Andrew; Mottram, Rachel; Whetton, Anthony D

    2003-09-01

    The H-2Kappab temperature-sensitive (ts) A58 transgenic (Immorto) mouse has been used previously to generate conditionally immortalized cells from a number of tissues. The present study aimed to investigate characteristics of primitive myeloid precursor cells derived from H-2Kappab-tsA58 bone marrow. Cell populations were enriched for granulocyte/macrophage progenitors by centrifugal elutriation, and were cultured in the presence and absence of cytokines at the permissive and restrictive temperatures for the A58 oncogene. Cells derived from H-2Kappab-tsA58 mice required both A58 activation and the growth factors, stem cell factor (SCF) and interleukin-3 (IL-3), for long-term cell survival and growth; cells were maintained for > 300 d in culture under these conditions. IL-3- and SCF-dependent clonal cell lines were derived with a phenotype (lin-, Sca-1+, CD34+, ER-MP 58+, ER-MP 12+, ER-MP 20-) characteristic of primitive myeloid progenitors. These cells differentiated on addition of granulocyte/macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF) and acquired mature cell morphology with some upregulation of differentiation markers. In conclusion, the A58 oncogene can immortalize haemopoietic progenitor cells. These cells require two cytokines for growth, IL-3 and SCF; as such, they constitute a useful resource for the study of synergistic interactions between growth factors. The ability to develop monocytic cell characteristics also permits the investigation of cytokine-mediated early haemopoietic progenitor cell development.

  8. A trial of alemtuzumab adjunctive therapy in allogeneic hematopoietic cell transplantation with minimal conditioning for severe combined immunodeficiency.

    PubMed

    Dvorak, Christopher C; Horn, Biljana N; Puck, Jennifer M; Adams, Stuart; Veys, Paul; Czechowicz, Agnieszka; Cowan, Morton J

    2014-09-01

    For infants with SCID the ideal conditioning regimen before allogeneic HCT would omit cytotoxic chemotherapy to minimize short- and long-term complications. We performed a prospective pilot trial with alemtuzumab monotherapy to overcome NK-cell mediated immunologic barriers to engraftment. We enrolled four patients who received CD34-selected haploidentical cells, two of whom failed to engraft donor T cells. The two patients who engrafted had delayed T-cell reconstitution, despite rapid clearance of circulating alemtuzumab. Although well-tolerated, alemtuzumab failed to overcome immunologic barriers to donor engraftment. Furthermore, alemtuzumab may slow T-cell development in patients with SCID in the setting of a T-cell depleted graft.

  9. Nonmyeloablative Conditioning Regimen to Increase Engraftment of Gene-modified Hematopoietic Stem Cells in Young Rhesus Monkeys

    PubMed Central

    Tarantal, Alice F; Giannoni, Francesca; I Lee, C Chang; Wherley, Jennifer; Sumiyoshi, Teiko; Martinez, Michele; Kahl, Christoph A; Elashoff, David; Louie, Stan G; Kohn, Donald B

    2012-01-01

    Immune responses to transgene products may lead to rejection of transduced cells, limiting successful gene therapy for genetic diseases. While moderate dosages of chemotherapeutic agents such as busulfan may increase hematopoietic stem cells (HSC) engraftment, they are not immune suppressive and do not abrogate immune responses to transgene products. Studies focused on nonmyeloablative conditioning with busulfan ± fludarabine in a clinically relevant monkey model to induce immune suppression to allow cells expressing a foreign transgene product to persist. Bone marrow CD34+ HSC were transduced in two equal fractions using simian immunodeficiency virus (SIV)-based lentiviral vectors carrying a nonexpressed DNA sequence tag (NoN) and the green fluorescent protein (GFP) reporter gene. Post-transplant there was no evidence of elimination of cells containing the potentially immunogenic GFP gene; several recipients had stable persistence of cells, and no differences were detected with fludarabine, which was rapidly cleared. Antibodies and cellular immune responses to GFP developed in recipients with the highest levels of GFP-marked cells, although these cells were not eliminated. These studies establish a clinically relevant pediatric primate model to assess the effects of conditioning regimens on the engraftment of transduced HSC and the immune responses to cells expressing a foreign gene product. PMID:22294147

  10. Regulatory role of hexosamine biosynthetic pathway on hepatic cancer stem cell marker CD133 under low glucose conditions

    NASA Astrophysics Data System (ADS)

    Lin, Shu-Hai; Liu, Tengfei; Ming, Xiaoyan; Tang, Zhi; Fu, Li; Schmitt-Kopplin, Philippe; Kanawati, Basem; Guan, Xin-Yuan; Cai, Zongwei

    2016-02-01

    Cancer was hypothesized to be driven by cancer stem cells (CSCs), but the metabolic determinants of CSC-like phenotype still remain elusive. Here, we present that hexosamine biosynthetic pathway (HBP) at least in part rescues cancer cell fate with inactivation of glycolysis. Firstly, metabolomic analysis profiled cellular metabolome in CSCs of hepatocellular carcinoma using CD133 cell-surface marker. The metabolic signatures of CD133-positive subpopulation compared to CD133-negative cells highlighted HBP as one of the distinct metabolic pathways, prompting us to uncover the role of HBP in maintenance of CSC-like phenotype. To address this, CSC-like phenotypes and cell survival were investigated in cancer cells under low glucose conditions. As a result, HBP inhibitor azaserine reduced CD133-positive subpopulation and CD133 expression under high glucose condition. Furthermore, treatment of N-Acetylglucosamine in part restores CD133-positive subpopulation when either 2.5 mM glucose in culture media or glycolytic inhibitor 2-deoxy-D-glucose in HCC cell lines was applied, enhancing CD133 expression as well as promoting cancer cell survival. Together, HBP might be a key metabolic determinant in the functions of hepatic CSC marker CD133.

  11. Unresolved issues in hematopoietic stem cell transplantation for severe combined immunodeficiency: need for safer conditioning and reduced late effects.

    PubMed

    Horn, Biljana; Cowan, Morton J

    2013-05-01

    In this review we discuss recent outcomes of hematopoietic cell transplantation (HCT) for patients with severe combined immunodeficiency (SCID), including survival, T- and B-cell reconstitution, and late effects, particularly those related to genotype, use of conditioning regimen, and use of alternative donors. We identify the following issues that require additional data, which can be obtained through cooperative studies: outcomes of patients with SCID who did not receive conditioning before alternative donor HCT; outcomes of patients with SCID who did not receive graft-versus-host disease prophylaxis after T cell-replete HCT; late effects of HCT for patients with SCID, including neurocognitive outcomes, growth, and development; and their relationship to genotype and use of alkylating agents for conditioning. Careful follow-up of outcomes of all newborns receiving diagnoses based on newborn screening programs for SCID is essential because data are scarce on the effects of conditioning regimens in very young patients. A consensus on the definition of T- and B-cell recovery, criteria for additional "boosts," pharmacokinetic data of chemotherapy agents used in young children, and uniformity of the use of various chemotherapy agents are needed to compare results among institutions. Finally, development of new nontoxic conditioning regimens for HCT that can be safely used in very young children is required.

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

    PubMed

    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.

  13. High-Performance Fully Printable Perovskite Solar Cells via Blade-Coating Technique under the Ambient Condition

    SciTech Connect

    Yang, Zhibin; Chueh, Chu-Chen; Zuo, Fan; Kim, Jong H.; Liang, Po-Wei; Jen, Alex K. -Y.

    2015-04-30

    A fully printable perovskite solar cell (PVSC) is demonstrated using a blade-coating technique under ambient conditions with controlled humidity. The influence of humidity on perovskite's crystallization is systematically investigated to realize the ambient processing condition. A high power conversion efficiency of 10.44% is achieved after optimizing the blade-coating process and, more importantly, a high-performance flexible PVSC is demonstrated for the first time. A high efficiency of 7.14% is achieved.

  14. A facile method to establish human induced pluripotent stem cells from adult blood cells under feeder-free and xeno-free culture conditions: a clinically compliant approach.

    PubMed

    Chou, Bin-Kuan; Gu, Haihui; Gao, Yongxing; Dowey, Sarah N; Wang, Ying; Shi, Jun; Li, Yanxin; Ye, Zhaohui; Cheng, Tao; Cheng, Linzhao

    2015-04-01

    Reprogramming human adult blood mononuclear cells (MNCs) cells by transient plasmid expression is becoming increasingly popular as an attractive method for generating induced pluripotent stem (iPS) cells without the genomic alteration caused by genome-inserting vectors. However, its efficiency is relatively low with adult MNCs compared with cord blood MNCs and other fetal cells and is highly variable among different adult individuals. We report highly efficient iPS cell derivation under clinically compliant conditions via three major improvements. First, we revised a combination of three EBNA1/OriP episomal vectors expressing five transgenes, which increased reprogramming efficiency by ≥10-50-fold from our previous vectors. Second, human recombinant vitronectin proteins were used as cell culture substrates, alleviating the need for feeder cells or animal-sourced proteins. Finally, we eliminated the previously critical step of manually picking individual iPS cell clones by pooling newly emerged iPS cell colonies. Pooled cultures were then purified based on the presence of the TRA-1-60 pluripotency surface antigen, resulting in the ability to rapidly expand iPS cells for subsequent applications. These new improvements permit a consistent and reliable method to generate human iPS cells with minimal clonal variations from blood MNCs, including previously difficult samples such as those from patients with paroxysmal nocturnal hemoglobinuria. In addition, this method of efficiently generating iPS cells under feeder-free and xeno-free conditions allows for the establishment of clinically compliant iPS cell lines for future therapeutic applications.

  15. A Facile Method to Establish Human Induced Pluripotent Stem Cells From Adult Blood Cells Under Feeder-Free and Xeno-Free Culture Conditions: A Clinically Compliant Approach

    PubMed Central

    Chou, Bin-Kuan; Gu, Haihui; Gao, Yongxing; Dowey, Sarah N.; Wang, Ying; Shi, Jun; Li, Yanxin; Ye, Zhaohui; Cheng, Tao

    2015-01-01

    Reprogramming human adult blood mononuclear cells (MNCs) cells by transient plasmid expression is becoming increasingly popular as an attractive method for generating induced pluripotent stem (iPS) cells without the genomic alteration caused by genome-inserting vectors. However, its efficiency is relatively low with adult MNCs compared with cord blood MNCs and other fetal cells and is highly variable among different adult individuals. We report highly efficient iPS cell derivation under clinically compliant conditions via three major improvements. First, we revised a combination of three EBNA1/OriP episomal vectors expressing five transgenes, which increased reprogramming efficiency by ≥10–50-fold from our previous vectors. Second, human recombinant vitronectin proteins were used as cell culture substrates, alleviating the need for feeder cells or animal-sourced proteins. Finally, we eliminated the previously critical step of manually picking individual iPS cell clones by pooling newly emerged iPS cell colonies. Pooled cultures were then purified based on the presence of the TRA-1-60 pluripotency surface antigen, resulting in the ability to rapidly expand iPS cells for subsequent applications. These new improvements permit a consistent and reliable method to generate human iPS cells with minimal clonal variations from blood MNCs, including previously difficult samples such as those from patients with paroxysmal nocturnal hemoglobinuria. In addition, this method of efficiently generating iPS cells under feeder-free and xeno-free conditions allows for the establishment of clinically compliant iPS cell lines for future therapeutic applications. PMID:25742692

  16. Surface-engineered substrates for improved human pluripotent stem cell culture under fully defined conditions.

    PubMed

    Saha, Krishanu; Mei, Ying; Reisterer, Colin M; Pyzocha, Neena Kenton; Yang, Jing; Muffat, Julien; Davies, Martyn C; Alexander, Morgan R; Langer, Robert; Anderson, Daniel G; Jaenisch, Rudolf

    2011-11-15

    The current gold standard for the culture of human pluripotent stem cells requires the use of a feeder layer of cells. Here, we develop a spatially defined culture system based on UV/ozone radiation modification of typical cell culture plastics to define a favorable surface environment for human pluripotent stem cell culture. Chemical and geometrical optimization of the surfaces enables control of early cell aggregation from fully dissociated cells, as predicted from a numerical model of cell migration, and results in significant increases in cell growth of undifferentiated cells. These chemically defined xeno-free substrates generate more than three times the number of cells than feeder-containing substrates per surface area. Further, reprogramming and typical gene-targeting protocols can be readily performed on these engineered surfaces. These substrates provide an attractive cell culture platform for the production of clinically relevant factor-free reprogrammed cells from patient tissue samples and facilitate the definition of standardized scale-up friendly methods for disease modeling and cell therapeutic applications.

  17. Direct production of organic acids from starch by cell surface-engineered Corynebacterium glutamicum in anaerobic conditions

    PubMed Central

    2013-01-01

    We produced organic acids, including lactate and succinate, directly from soluble starch under anaerobic conditions using high cell-density cultures of Corynebacterium glutamicum displaying α-amylase (AmyA) from Streptococcus bovis 148 on the cell surface. Notably, reactions performed under anaerobic conditions at 35 and 40°C, which are higher than the optimal growth temperature of 30°C, showed 32% and 19%, respectively, higher productivity of the organic acids lactate, succinate, and acetate compared to that at 30°C. However, α-amylase was not stably anchored and released into the medium from the cell surface during reactions at these higher temperatures, as demonstrated by the 61% and 85% decreases in activity, respectively, from baseline, compared to the only 8% decrease at 30°C. The AmyA-displaying C. glutamicum cells retained their starch-degrading capacity during five 10 h reaction cycles at 30°C, producing 107.8 g/l of total organic acids, including 88.9 g/l lactate and 14.0 g/l succinate. The applicability of cell surface-engineering technology for the production of organic acids from biomass by high cell-density cultures of C. glutamicum under anaerobic conditions was demonstrated. PMID:24342107

  18. [Differentiated conditions for the induction of suberin synthesis or cell proliferation in potato tuber tissue after derepression].

    PubMed

    Lange, H; Rosenstock, G; Kahl, G

    1970-06-01

    Experiments with potato tuber fragments under normal atmospheric conditions (0.03% CO2) suggest that a wound periderm, prominently marked by suberin synthesis, develops regularly, independently of preparation technics. It has been demonstrated that potato parenchym is able to perform differentiated reactions depending on the changing influences on the reacting cells caused by the wound stimulus and partial isolation. Suberin synthesis and the cicatrization effect can be suppressed totally if changes remain insignificant as compared with the cellular state in the intact organ. This requires: 1. separating the tissue without bruising it to avoid deformations of the protoplasmatic structure; 2. removing adjacent fragments of destroyed cells by rinsing the slices; 3. maintaining cell turgidity by keeping the moisture in the reaction atmosphere at 100%. Moreover 10% CO2-concentration is necessary, corresponding to the intercellular air in situ.Whithin 24 hours after depression these conditions induce spontaneous proliferation uniformly in all superficial cell layers of tissue slices, accompanied by multiple mitosis in place of the typical suberized wound periderm. Inhibition of suberin biosynthesis is reversible by subsequent modification of reaction conditions. In contrast a method has been found to induce suberin formation only and to suppress any cell division activity (Kahl et al., 1969 a). This provides the opportunity to analyse causal corresponding physiological reactions in genetically identical cells. The advantage of this method-compared with long term experiments involving hormone-activated growth in sterile tissue culture - is the possibility of controlling quantitative and qualitative changes in biochemical pathways immediately after derepression.

  19. Mesenchymal stem cell-conditioned medium accelerates skin wound healing: An in vitro study of fibroblast and keratinocyte scratch assays

    SciTech Connect

    Walter, M.N.M.; Wright, K.T.; Fuller, H.R.; MacNeil, S.; Johnson, W.E.B.

    2010-04-15

    We have used in vitro scratch assays to examine the relative contribution of dermal fibroblasts and keratinocytes in the wound repair process and to test the influence of mesenchymal stem cell (MSC) secreted factors on both skin cell types. Scratch assays were established using single cell and co-cultures of L929 fibroblasts and HaCaT keratinocytes, with wound closure monitored via time-lapse microscopy. Both in serum supplemented and serum free conditions, wound closure was faster in L929 fibroblast than HaCaT keratinocyte scratch assays, and in co-culture the L929 fibroblasts lead the way in closing the scratches. MSC-CM generated under serum free conditions significantly enhanced the wound closure rate of both skin cell types separately and in co-culture, whereas conditioned medium from L929 or HaCaT cultures had no significant effect. This enhancement of wound closure in the presence of MSC-CM was due to accelerated cell migration rather than increased cell proliferation. A number of wound healing mediators were identified in MSC-CM, including TGF-{beta}1, the chemokines IL-6, IL-8, MCP-1 and RANTES, and collagen type I, fibronectin, SPARC and IGFBP-7. This study suggests that the trophic activity of MSC may play a role in skin wound closure by affecting both dermal fibroblast and keratinocyte migration, along with a contribution to the formation of extracellular matrix.

  20. Determination of the optimum conditions for lung cancer cells treatment using cold atmospheric plasma

    NASA Astrophysics Data System (ADS)

    Akhlaghi, Morteza; Rajaei, Hajar; Mashayekh, Amir Shahriar; Shafiae, Mojtaba; Mahdikia, Hamed; Khani, Mohammadreza; Hassan, Zuhair Mohammad; Shokri, Babak

    2016-10-01

    Cold atmospheric plasmas (CAPs) can affect live cells and organisms due to the production of different reactive species. In this paper, the effects of various parameters of the CAP such as the treatment time, gas mixture, gas flow rate, applied voltage, and distance from the nozzle on the LL/2 lung cancer cell line have been studied. The probable effect of UV radiation has also been investigated using an MgF2 filter. Besides the cancerous cells, the 3T3 fibroblast cell line as a normal cell has been treated with the CAP. The Methylthiazol Tetrazolium assay showed that all parameters except the gas flow rate could impress effectively on the cancer cell viability. The cell proliferation seemed to be stopped after plasma treatment. The flow cytometry assay revealed that apoptosis and necrosis were appreciable. It was also found that treating time up to 2 min will not exert any effect on the normal cells.

  1. Altered morphology of hippocampal dentate granule cell presynaptic and postsynaptic terminals following conditional deletion of TrkB.

    PubMed

    Danzer, Steve C; Kotloski, Robert J; Walter, Cynthia; Hughes, Maya; McNamara, James O

    2008-01-01

    Dentate granule cells play a critical role in the function of the entorhinal-hippocampal circuitry in health and disease. Dentate granule cells are situated to regulate the flow of information into the hippocampus, a structure required for normal learning and memory. Correspondingly, impaired granule cell function leads to memory deficits, and, interestingly, altered granule cell connectivity may contribute to the hyperexcitability of limbic epilepsy. It is important, therefore, to understand the molecular determinants of synaptic connectivity of these neurons. Brain-derived neurotrophic factor and its receptor TrkB are expressed at high levels in the dentate gyrus (DG) of the hippocampus, and are implicated in regulating neuronal development, neuronal plasticity, learning, and the development of epilepsy. Whether and how TrkB regulates granule cell structure, however, is incompletely understood. To begin to elucidate the role of TrkB in regulating granule cell morphology, here we examine conditional TrkB knockout mice crossed to mice expressing green fluorescent protein in subsets of dentate granule cells. In stratum lucidum, where granule cell mossy fiber axons project, the density of giant mossy fiber boutons was unchanged, suggesting similar output to CA3 pyramidal cell targets. However, filopodial extensions of giant boutons, which contact inhibitory interneurons, were increased in number in TrkB knockout mice relative to wildtype controls, predicting enhanced feedforward inhibition of CA3 pyramidal cells. In knockout animals, dentate granule cells possessed fewer primary dendrites and enlarged dendritic spines, indicative of disrupted excitatory synaptic input to the granule cells. Together, these findings demonstrate that TrkB is required for development and/or maintenance of normal synaptic connectivity of the granule cells, thereby implying an important role for TrkB in the function of the granule cells and hippocampal circuitry.

  2. Defining the Operational Conditions for High Temperature Polymer Fuel Cells in Naval Environments

    DTIC Science & Technology

    2008-12-31

    benefits of both Proton Exchange Membrane Fuel Cells (PEMFCs) and phosphoric acid fuel cell technologies: a solid polymer electrolyte, the PBI ...PEMFC and phosphoric acid fuel cell technologies: a solid polymer electrolyte, the PBI membrane, but with higher temperature (160°C) operation. PBI ...high-temperature polymer fuel cell is emerging, based on phosphoric- acid -doped polybenzimidazole ( PBI ) membranes. PBI technology combines some of the

  3. An Investigation to Resolve the Interaction Between Fuel Cell, Power Conditioning System and Application Loads

    SciTech Connect

    Sudip K. Mazumder

    2005-12-31

    Development of high-performance and durable solidoxide fuel cells (SOFCs) and a SOFC power-generating system requires knowledge of the feedback effects from the power-conditioning electronics and from application-electrical-power circuits that may pass through or excite the power-electronics subsystem (PES). Therefore, it is important to develop analytical models and methodologies, which can be used to investigate and mitigate the effects of the electrical feedbacks from the PES and the application loads (ALs) on the reliability and performance of SOFC systems for stationary and non-stationary applications. However, any such attempt to resolve the electrical impacts of the PES on the SOFC would be incomplete unless one utilizes a comprehensive analysis, which takes into account the interactions of SOFC, PES, balance-of-plant system (BOPS), and ALs as a whole. SOFCs respond quickly to changes in load and exhibit high part- and full-load efficiencies due to its rapid electrochemistry, which is not true for the thermal and mechanical time constants of the BOPS, where load-following time constants are, typically, several orders of magnitude higher. This dichotomy can affect the lifetime and durability of the SOFCSs and limit the applicability of SOFC systems for load-varying stationary and transportation applications. Furthermore, without validated analytical models and investigative design and optimization methodologies, realizations of cost-effective, reliable, and optimal PESs (and power-management controls), in particular, and SOFC systems, in general, are difficult. On the whole, the research effort can lead to (a) cost-constrained optimal PES design for high-performance SOFCS and high energy efficiency and power density, (b) effective SOFC power-system design, analyses, and optimization, and (c) controllers and modulation schemes for mitigation of electrical impacts and wider-stability margin and enhanced system efficiency.

  4. Increased platelet adhesion under flow conditions is induced by both thalassemic platelets and red blood cells.

    PubMed

    Goldschmidt, Neta; Spectre, Galia; Brill, Alexander; Zelig, Orly; Goldfarb, Ada; Rachmilewitz, Eliezer; Varon, David

    2008-11-01

    Thromboembolic complications are not uncommon in thalassemia. Previous studies suggest increased platelet aggregation and a potential role of pathological changes in the red blood cell (RBC) lipid membrane, induced by oxidative stress. In the present study, platelet adhesion and the effect of thalassemic RBC on platelet adhesion under flow conditions were evaluated, using the Cone and Plate (let) Analyzer(CPA). Twenty-two beta-thalassemia patients and 22 blood type-matched healthy controls were studied. An increased platelet adhesion (% surface coverage, SC), was observed in patients as compared to controls (p < 0.05). When platelet count and haematocrit were normalized by autologous reconstitution, a significant increase in platelet aggregation (average size, AS) was observed (p < 0.05). Increased platelet adhesion (SC and AS), was demonstrated in six patients with a history of thrombosis as compared to 16 patients without any history of thrombosis (p < or = 0.007) and in 17 splenectomized patients as compared to five non-splenectomized patients (p = 0.003). In reconstitution studies, thalassemic RBC mixed with normal platelet-rich plasma significantly increased platelet adhesion compared to normal RBC (SC p < 0.03, AS p < 0.02). Thalassemic platelets reconstituted with normal RBC, had increased aggregation (AS, p < 0.004) in comparison with normal platelets. The results indicate that increased platelet adhesion in beta-thalassemia is induced by both platelets and RBC. Increased platelet adhesion correlated with clinical thrombotic events and thus may suggest a mechanism of thrombosis in thalassemic patients. The potential application of the CPA in identifying thalassemic patients with high risk for thrombosis should be studied prospectively in a larger cohort of patients.

  5. Investigating the neuroglial differentiation effect of neuroblastoma conditioned medium in human endometrial stem cells cultured on 3D nanofibrous scaffold.

    PubMed

    Ebrahimi-Barough, Somayeh; Hoveizi, Elham; Norouzi Javidan, Abbas; Ai, Jafar

    2015-08-01

    Neural tissue engineering is an important area of research in the field of tissue-engineering especially for neurodegenerative disease such as spinal cord injury. The differentiation capacity of human endometrial stem cells (hEnSCs) into neuronal cells has yet to be elucidated. Here, the major aim of the present study was to investigate the differentiation ability of hEnSCs cultured on polylactic acid/chitosan (PLA/CS) nanofibrous scaffold into neuroglial cells in response to conditioned medium of BE(2)-C human neuroblastoma cells and growth factors. Here we investigated the use PLA/CS scaffold as a three dimensional (3D) system that increased neuro-glial cells differentiation. Human EnSCs after three passages were differentiated in neuro-glial like cells under neuroblastoma conditioned medium with FGF2/PDGF-AA on PLA/CS scaffold. By day 18, differentiated cells were analyzed for expression of neuroglial markers by qRT-PCR and immunofluorescence. The results revealed that hEnSCs attach, grow and differentiation on the nanofibrous PLA/CS scaffold. Additionally, our study showed the expression of neural and glial lineage markers such as Nestin, NF-L, MAP2, PDGFRa, CNP, Olig2, MBP, and GFAP in the level of mRNA and MAP2, Tuj-1, and NF-L in the protein level after 18 days. Our results demonstrate that hEnSCs cultured on PLA/CS nanofibrous scaffold have the potential to differentiate in neuronal and glial cells in presence of neuroblastoma conditioned medium on PLA/CS scaffold. The result of this study may have impact in tissue engineering and cells-base therapy of neurodegenerative diseases and have a great potential for wide application.

  6. In vitro cementoblast-like differentiation of postmigratory neural crest-derived p75(+) stem cells with dental follicle cell conditioned medium.

    PubMed

    Wen, Xiujie; Liu, Luchuan; Deng, Manjing; Liu, Rui; Zhang, Li; Nie, Xin

    2015-09-10

    Cranial neural crest-derived cells (CNCCs) play important role in epithelial-mesenchymal interactions during tooth morphogenesis. However, the heterogeneity of CNCCs and their tendency to spontaneously differentiate along smooth muscle or osteoblast lineages in vitro limit further understanding of their biological properties. We studied the differentiation properties of isolated rat embryonic postmigratory CNCCs, expressing p75 neurotrophin receptor (p75NTR). These p75NTR positive (p75(+)) CNCCs, isolated using fluorescence activated cell sorter, exhibited fibroblast-like morphology and characteristics of mesenchymal stem cells. Incubation of p75(+) CNCCs in dental follicle cell conditioned medium (DFCCM) combined with dentin non-collagenous proteins (dNCPs), altered their morphological features to cementoblast-like appearance. These cells also showed low proliferative activity, high ALP activity and significantly increased calcified nodule formation. Markers related to mineralization or specific to cementoblast lineage were highly expressed in dNCPs/DFCCM-treated p75(+) cells, suggesting their differentiation along cementoblast-like lineage. p75(+) stem cells selected from postmigratory CNCCs represent a pure stem cell population and could be used as a stem cell model for in vitro studies due to their intrinsic ability to differentiate to neuronal cells and transform from neuroectoderm to ectomesenchyme. They can provide a potential stem cell resource for tooth engineering studies and help to further investigate mechanisms of epithelial-mesenchymal interactions in tooth morphogenesis.

  7. Activity of interferon-dependent 2',5'-oligoadenylate synthetase in rat lymphoid cells under transformed environment conditions

    NASA Astrophysics Data System (ADS)

    Ostapchenko, L. I.; Mikhailik, I. V.; Prokopova, K. V.

    It is detected that interferon-dependent 2',5'-oligoadenylate synthetase is a sensitive index of immunocompetent cells functional state under transformed environment conditions. Microgravitation and ionising radiation induce increase of investigated enzyme activity in rat lymphocytes, which can be a result of lymphoid cells compensatory mechanisms starting in response to stress factors action. Administration of interferon inductors permits to stimulate the 2',5'-oligoadenylate synthetase, which enables one to correct pathological changes in the cells and to intensify adaptive reactions of immune systems.

  8. PD-1 expression conditions T cell avidity within an antigen-specific repertoire

    PubMed Central

    Simon, Sylvain; Vignard, Virginie; Florenceau, Laetitia; Dreno, B.; Khammari, A.; Lang, F.; Labarriere, N.

    2016-01-01

    ABSTRACT Despite its negative regulatory role on tumor-specific T cells, Programmed cell death 1 (PD-1) is also a marker of activated tumor-infiltrating T cells. In cancer, PD-1 blockade partially reverses T cell dysfunction allowing the amplification of tumor reactive T cells. Here, we investigated the role of PD-1 signaling on effector/memory human T cells specific for shared melanoma antigens, derived from blood. We documented for the first time the existence of melanoma-specific T cell clones unable to express PD-1. This stable feature was due to the persistent methylation of the PDCD1 promoter. These PD-1neg clones were of lower avidity than their PD-1pos counterparts, suggesting that high-affinity-specific T cell clones unable to express PD-1 are not or rarely present in peripheral blood, as they are probably eliminated by negative selection, due to their high reactivity. We also documented the existence of such PD-1neg T cell clones in melanoma tumor-infiltrating lymphocytes (TIL), which also exhibited a lower functional avidity than PD-1pos TIL clones. This clearly shows that PD-1 expression identifies antigen-specific T cell clonotypes of high functional avidity. Finally, we demonstrated that PD-1 blockade during the in vitro selection process of Melan-A-specific T cells favored the amplification of higher avidity T cell clonotypes. This preferential amplification of high-avidity memory T cells upon PD-1 blockade resonates with the expansion of reactive T cells, including neo-antigen-specific T cells observed in anti-PD-1-treated patients. This feature should also be a useful biomarker of clinical efficiency, while providing new insights for adoptive transfer treatments. PMID:26942093

  9. Efficient procedure for grapevine embryogenic suspension establishment and plant regeneration: role of conditioned medium for cell proliferation.

    PubMed

    Ben Amar, A; Cobanov, P; Boonrod, K; Krczal, G; Bouzid, S; Ghorbel, A; Reustle, G M

    2007-09-01

    An efficient system for the establishment and multiplication of highly prolific embryogenic cell cultures of grapevine (Vitis sp.) was developed. Using anther-derived pro-embryogenic masses as starting material, cell suspensions of different grapevine cultivars (Tempranillo, Cabernet-Sauvignon) and rootstocks (Kober 125 AA, Kober 5 BB, 110 Richter) were initiated in liquid medium containing NOA (1.0 mg l(-1)) and BAP (0.25 mg l(-1)) as growth regulators. Conditioned medium was recovered and utilised for establishing new, highly totipotent cell cultures. The suspensions obtained, showed embryogenic competence resulting in somatic embryo induction and subsequent plant regeneration. In this study, a simplified establishment procedure for grapevine embryogenic cell suspension allowing the fast multiplication of embryogenic material is described. Evidence for the promoting effect of the protein fraction derived from conditioned medium, on cell proliferation was found. In bioassays, addition of ss-D: -GlcY affect cell proliferation suggesting that arabinogalactan proteins are required for growth processes in grapevine cell cultures.

  10. Conditional control of dendritic cell factor 1 expression by a tetracycline-inducible system.

    PubMed

    Yan, H; Huang, C; Yang, M; Guo, J; Wang, J; Feng, R; Wen, T

    2015-05-08

    Dendritic cell factor 1 plays important roles in neural stem cells differentiation and in glioma cells proliferation, migration, and invasion. Here, we used a tetracycline—inducible system that regulates the expression of Dendritic cell factor 1 in glioma cells. We constructed two tet—inducible vectors, pTRE—EGFP—DCF1 and pTRE—LJM1—DCF1, by modifying the promoter PCMV. In the absence of tetracycline or doxycycline, the expression of Dendritic cell factor 1 in cells co—transfected with pTRE—EGFP—DCF1 or pTRE—LJM1—EGFP—DCF1 and ptTS—Neo was suppressed through binding of the tetracyline—controlled transcriptional suppressor to tetracycline response element, and the suppression was released by the addition of doxycycline. Our work has laid foundations for potential clinical application of cancer therapy in realizing artificial regulation of gene.

  11. Establishment and growth responses of Nile tilapia embryonic stem-like cell lines under feeder-free condition.

    PubMed

    Fan, Zhenhua; Liu, Linyan; Huang, Xiaohuan; Zhao, Yang; Zhou, Linyan; Wang, Deshou; Wei, Jing

    2017-02-01

    Embryonic stem (ES) cells provide an invaluable tool for molecular analysis of vertebrate development and a bridge linking genomic manipulations in vitro and functional analysis of target genes in vivo. Work towards fish ES cells so far has focused on zebrafish (Danio renio) and medaka (Oryzias latipes). Here we describe the derivation, pluripotency, differentiation and growth responses of ES cell lines from Nile tilapia (Oreochromis niloticus), a world-wide commercial farmed fish. These cell lines, designated as TES1-3, were initiated from blastomeres of Nile tilapia middle blastula embryos (MBE). One representative line, TES1, showed stable growth and phenotypic characteristics of ES cells over 200 days of culture with more than 59 passages under feeder-free conditions. They exhibited high alkaline phosphatase activity and expression of pluripotency genes including pou5f3 (the pou5f1/oct4 homologue), sox2, myc and klf4. In suspension culture together with retinoic acid treatment, TES1 cells formed embryoid bodies, which exhibited expression profile of differentiation genes characteristics of all three germ cell layers. Notably, PKH26-labeled TES1 cells introduced into Nile tilapia MBE could contribute to body compartment development and led to hatched chimera formation with an efficacy of 13%. These results suggest that TES1 cells have pluripotency and differentiation potential in vitro and in vivo. In the conditioned DMEM, all of the supplements including the fetal bovine serum, fish embryonic extract, fish serum, basic fibroblast growth factor and non-protein supplement combination 5N were mitogenic for TES1 cell growth. This study will promote ES-based biotechnology in commercial fish.

  12. The dietary flavonoid kaempferol effectively inhibits HIF-1 activity and hepatoma cancer cell viability under hypoxic conditions

    SciTech Connect

    Mylonis, Ilias; Lakka, Achillia; Tsakalof, Andreas; Simos, George

    2010-07-16

    Research highlights: {yields} Kaempferol inhibits HIF-1 activity in hepatocarcinoma cells; {yields} Kaempferol causes cytoplasmic mislocalization of HIF-1{alpha} by impairing the MAPK pathway. {yields} Viability of hepatocarcinoma cells under hypoxia is reduced by kaempferol. -- Abstract: Hepatocellular carcinoma (HCC) is characterized by high mortality rates and resistance to conventional treatment. HCC tumors usually develop local hypoxia, which stimulates proliferation of cancer cells and renders them resilient to chemotherapy. Adaptation of tumor cells to the hypoxic conditions depends on the hypoxia-inducible factor 1 (HIF-1). Over-expression of its regulated HIF-1{alpha} subunit, an important target of anti-cancer therapy, is observed in many cancers including HCC and is associated with severity of tumor growth and poor patient prognosis. In this report we investigate the effect of the dietary flavonoid kaempferol on activity, expression levels and localization of HIF-1{alpha} as well as viability of human hepatoma (Huh7) cancer cells. Treatment of Huh7 cells with kaempferol under hypoxic conditions (1% oxygen) effectively inhibited HIF-1 activity in a dose-dependent manner (IC{sub 50} = 5.16 {mu}M). The mechanism of this inhibition did not involve suppression of HIF-1{alpha} protein levels but rather its mislocalization into the cytoplasm due to inactivation of p44/42 MAPK by kaempferol (IC{sub 50} = 4.75 {mu}M). Exposure of Huh7 cells to 10 {mu}{Mu} kaempferol caused significant reduction of their viability, which was remarkably more evident under hypoxic conditions. In conclusion, kaempferol, a non-toxic natural food component, inhibits both MAPK and HIF-1 activity at physiologically relevant concentrations (5-10 {mu}M) and suppresses hepatocarcinoma cell survival more efficiently under hypoxia. It has, therefore, potential as a therapeutic or chemopreventive anti-HCC agent.

  13. Conditional and specific NF-kappaB blockade protects pancreatic beta cells from diabetogenic agents.

    PubMed

    Eldor, R; Yeffet, A; Baum, K; Doviner, V; Amar, D; Ben-Neriah, Y; Christofori, G; Peled, A; Carel, J C; Boitard, C; Klein, T; Serup, P; Eizirik, D L; Melloul, D

    2006-03-28

    Type 1 diabetes is characterized by the infiltration of inflammatory cells into pancreatic islets of Langerhans, followed by the selective and progressive destruction of insulin-secreting beta cells. Islet-infiltrating leukocytes secrete cytokines such as IL-1beta and IFN-gamma, which contribute to beta cell death. In vitro evidence suggests that cytokine-induced activation of the transcription factor NF-kappaB is an important component of the signal triggering beta cell apoptosis. To study the in vivo role of NF-kappaB in beta cell death, we generated a transgenic mouse line expressing a degradation-resistant NF-kappaB protein inhibitor (DeltaNIkappaBalpha), acting specifically in beta cells, in an inducible and reversible manner, by using the tet-on regulation system. In vitro, islets expressing the DeltaNIkappaBalpha protein were resistant to the deleterious effects of IL-1beta and IFN-gamma, as assessed by reduced NO production and beta-cell apoptosis. This effect was even more striking in vivo, where nearly complete protection against multiple low-dose streptozocin-induced diabetes was observed, with reduced intraislet lymphocytic infiltration. Our results show in vivo that beta cell-specific activation of NF-kappaB is a key event in the progressive loss of beta cells in diabetes. Inhibition of this process could be a potential effective strategy for beta-cell protection.

  14. Analysis of thymic stromal cell subpopulations grown in vitro on extracellular matrix in defined medium. I. Growth conditions and morphology of murine thymic epithelial and mesenchymal cells.

    PubMed

    Eshel, I; Savion, N; Shoham, J

    1990-03-01

    We report here the successful selective cultivation of murine thymic mesenchymal reticular cells (MTMC) and murine thymic epithelial cells (MTEC) grown on extracellular matrix in the presence of defined medium. The selective growth of these two cell types was based on 1) conditions of tissue disruption and 2) differential growth requirements. Both cell types were dependent on transferrin, high density lipoproteins, insulin, hydrocortisone, and epidermal growth factor, whereas MTMC was dependent also on selenium and 3,5,3'-triiodothyronine. The elimination of single factors or extracellular matrix resulted in specific and different changes in the growth pattern of each cell subpopulation. Cells of both types exhibited the ultrastructural features of high metabolic activity. The epithelial nature of MTEC cultures was defined by bundles of tonofilaments and desmosomes and by positive staining to keratins and negative to vimentin. In addition MTEC were positively stained with mAb to thymic medullary epithelial cells and by Ulex europeus agglutinin, and were able to form Hassall's corpuscles, suggesting their medullary origin. MTEC were also H-2 and Ia positive. In contrast MTMC were positive for vimentin and periodic acid-Schiff, low positive for H-2, and negative for keratin and Ia. Both cells did not contain nonspecific esterase, nor did they phagocytize latex beads. With the use of all these criteria we classified MTEC as epithelial cells from the medullary compartment of the thymus and MTMC as reticular cells of mesenchymal origin.

  15. Genetic stability of murine pluripotent and somatic hybrid cells may be affected by conditions of their cultivation.

    PubMed

    Ivanovna, Shramova Elena; Alekseevich, Larionov Oleg; Mikhailovich, Khodarovich Yurii; Vladimirovna, Zatsepina Olga

    2011-01-01

    Using mouse pluripotent teratocarcinoma PCC4azal cells and proliferating spleen lymphocytes we obtained a new type of hybrids, in which marker lymphocyte genes were suppressed, but expression the Oct-4 gene was not effected; the hybrid cells were able to differentiate to cardiomyocytes. In order to specify the environmental factors which may affect the genetic stability and other hybrid properties, we analyzed the total chromosome number and differentiation potencies of hybrids respectively to conditions of their cultivation. Particular attention was paid to the number and transcription activity of chromosomal nucleolus organizing regions (NORs), which harbor the most actively transcribed - ribosomal - genes. The results showed that the hybrids obtained are characterized by a relatively stable chromosome number which diminished less than in 5% during 27 passages. However, a long-term cultivation of hybrid cells in non-selective conditions resulted in preferential elimination of some NO- chromosomes, whereas the number of active NORs per cell was increased due to activation of latent NORs. On the contrary, in selective conditions, i.e. in the presence of hypoxantine, aminopterin and thymidine, the total number of NOR-bearing chromosomes was not changed, but a partial inactivation of remaining NORs was observed. The higher number of active NORs directly correlated with the capability of hybrid cells for differentiation to cardiomyocytes.

  16. Derivation of embryonic stem cells from Kunming mice IVF blastocyst in feeder- and serum-free condition.

    PubMed

    Liu, Xiaokun; Wei, Qiang; Zhang, Junhong; Yang, Wanli; Zhao, Xiaoe; Ma, Baohua

    2015-06-01

    Kunming mice are widely used in China; however, it is difficult to isolate embryonic stem cells (ESCs) in conventional derivation condition containing feeder cells and serum. 6-Bromoindirubin-3'-oxime (BIO), a glycogen synthase kinase 3 (GSK3) inhibitor, could facilitate the maintenance of pluripotency of ESCs. Therefore, BIO could be considered as a candidate to replace feeder cells and serum. On the other hand, in vitro fertilization (IVF) is an important technology in assisted reproduction. It is reported that there was some difference in gene expression between IVF and in vivo developed blastocyst. ESCs derived from IVF blastocyst could provide a valuable tool to research the effect of IVF on differentiation and development. In the present study, we established two novel ESC lines from IVF blastocyst of Kunming mice in a feeder- and serum-free condition containing 2.5 μM BIO. In this condition, expanded IVF blastocyst could spontaneously hatch from zonae pellucidae and attached to the gelatin-coated bottom of dishes. ESC-like outgrowth could be observed without overfull trophoblast cells. After further propagation, two Kunming mice ESC lines, designated as KMES1 and KMES2, were obtained. These two novel ESCs shared common morphological characteristics with other rodent ESCs, showed strong alkaline phosphatase activity, and expressed pluripotent markers, including Oct-4, Nanog, and SSEA-1. Embryoid body (EB) and teratoma test indicated that these ESCs could spontaneously differentiate into cells representative of all three embryonic germ layers.

  17. Depletion of host CCR7(+) dendritic cells prevented donor T cell tissue tropism in anti-CD3-conditioned recipients.

    PubMed

    He, Wei; Racine, Jeremy J; Johnston, Heather F; Li, Xiaofan; Li, Nainong; Cassady, Kaniel; Liu, Can; Deng, Ruishu; Martin, Paul; Forman, Stephen; Zeng, Defu

    2014-07-01

    We reported previously that anti-CD3 mAb treatment before hematopoietic cell transplantation (HCT) prevented graft-versus-host disease (GVHD) and preserved graft-versus-leukemia (GVL) effects in mice. These effects were associated with downregulated donor T cell expression of tissue-specific homing and chemokine receptors, marked reduction of donor T cell migration into GVHD target tissues, and deletion of CD103(+) dendritic cells (DCs) in mesenteric lymph nodes (MLN). MLN CD103(+) DCs and peripheral lymph node (PLN) DCs include CCR7(+) and CCR7(-) subsets, but the role of these DC subsets in regulating donor T cell expression of homing and chemokine receptors remain unclear. Here, we show that recipient CCR7(+), but not CCR7(-), DCs in MLN induced donor T cell expression of gut-specific homing and chemokine receptors in a retinoid acid-dependent manner. CCR7 regulated activated DC migration from tissue to draining lymph node, but it was not required for the ability of DCs to induce donor T cell expression of tissue-specific homing and chemokine receptors. Finally, anti-CD3 treatment depleted CCR7(+) but not CCR7(-) DCs by inducing sequential expansion and apoptosis of CCR7(+) DCs in MLN and PLN. Apoptosis of CCR7(+) DCs was associated with DC upregulation of Fas expression and natural killer cell but not T, B, or dendritic cell upregulation of FasL expression in the lymph nodes. These results suggest that depletion of CCR7(+) host-type DCs, with subsequent inhibition of donor T cell migration into GVHD target tissues, can be an effective approach in prevention of acute GVHD and preservation of GVL effects.

  18. Cell density-correlated induction of pyruvate decarboxylase under aerobic conditions in the yeast Pichia stipitis.

    PubMed

    Mergler, M; Klinner, U

    2001-01-01

    During the aerobic batch cultivation of P. stipitis CBS 5776 with glucose, pyruvate decarboxylase was activated in a cell number-correlated manner. Activation started when a cell number between 7 x 10(7) and x 10(8) cells ml(-1) was reached and the enzyme activity increased during further cultivation. This induction might have been triggered either by an unknown quorum sensing system or by a shortage of cytoplasmic acetyl-CoA.

  19. The effects of anti-vascular endothelial growth factor agents on human retinal pigment epithelial cells under high glucose conditions

    PubMed Central

    Oh, Jong Rok; Han, Jung Woo; Kim, Yoon Kyung; Ohn, Young-Hoon; Park, Tae Kwann

    2017-01-01

    AIM To investigate the effects of high glucose levels and anti-vascular endothelial growth factor (VEGF) agents (bevacizumab, ranibizumab and aflibercept) on retinal pigment epithelium (RPE) cells. METHODS ARPE-19 cells were cultured at different glucose levels (5.5 mmol/L, 25 mmol/L, and 75 mmol/L). Cell viability was evaluated by MTT assay at 3d after treatment with D-glucose. Cell migration ability was measured by wound healing assay at 3d. A cell death detection kit was used to assess apoptosis at 3 and 14d. Cell proliferation was assessed by EdU assay at 3d. The culture medium was treated with anti-VEGF agents at clinically relevant concentrations. The experiment was then repeated at a different glucose level. RESULTS The viability and migration of ARPE-19 cells were significantly decreased in the presence of 75 mmol/L as compared to 5.5 mmol/L glucose. The percentage of TUNEL-positive cells was significantly increased and the proliferative potential was decreased with 75 mmol/L compared to 5.5 mmol/L glucose. There were no significant differences in the results between 25 mmol/L and 5.5 mmol/L glucose. In the presence of 75 mmol/L glucose, the groups treated with anti-VEGF showed decreased cell viability and proliferation and increased apoptosis. However, there were no significant differences between the anti-VEGF groups. CONCLUSION High glucose level decreases the viability, wound healing ability, and proliferation of RPE cells, while increasing apoptosis. Furthermore, anti-VEGF agents interfered with the physiological functions of RPE cells under high-glucose conditions, accompanied by decreases in cell viability and proliferation. PMID:28251077

  20. Method for Producing Non-Neoplastic, Three Dimensional, Mammalian Tissue and Cell Aggregates Under Microgravity Culture Conditions and the Products Produced Therefrom

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas J. (Inventor); Wolf, David A. (Inventor); Spaulding, Glenn F. (Inventor); Prewett, Tracey L. (Inventor)

    1996-01-01

    Normal mammalian tissue and the culturing process has been developed for the three groups of organ, structural, and blood tissue. The cells are grown in vitro under microgravity culture conditions and form three dimensional cells aggregates with normal cell function. The microgravity culture conditions may be microgravity or simulated microgravity created in a horizontal rotating wall culture vessel.

  1. CLA reduces inflammatory mediators from A427 human lung cancer cells and A427 conditioned medium promotes differentiation of C2C12 murine muscle cells.

    PubMed

    Oraldi, Manuela; Maggiora, Marina; Paiuzzi, Elena; Canuto, Rosa A; Muzio, Giuliana

    2013-01-01

    Conjugated linoleic acid (CLA) is thought to have anti-proliferative and anti-inflammatory properties, but its effect on cancer cachexia is unknown. Two effects were here investigated: that of CLA on inflammatory mediator production in human lung cancer cells, and that of reduced mediators on the myogenic differentiation of murine muscle C2C12 cells. The latter cells were grown in medium conditioned by human lung cancer A427 cells, with or without CLA, to mimic only the effect of molecules released from the tumor "in vivo", excluding the effect of host-produced cachectic factors. The results obtained show that CLA was found to reduce the production of tumor necrosis factor-α, interleukin (IL)-1β and prostaglandin E2 (PGE2), but had no effect on IL-6 production. The mechanisms underlying the effect of CLA on cytokine or PGE2 release in A427 cells are probably mediated by activation of peroxisome proliferator-activated receptor (PPAR)α, which increased at 24 h CLA treatment. In turn, the reduced content of inflammatory mediators in medium conditioned by A427 cells, in the presence of CLA, allowed muscle cells to proliferate, again by inducing PPAR. The involvement of PPARα was demonstrated by treatment with the antagonist MK-886. The findings demonstrate the anti-inflammatory and myogenic action of CLA and point to its possible application as a novel dietary supplement and therapeutic agent in inflammatory disease states, such as cachexia.

  2. Adaptation of Human Pluripotent Stem Cells to Feeder-Free Conditions in Chemically Defined Medium with Enzymatic Single-Cell Passaging

    PubMed Central

    Stover, Alexander E.; Schwartz, Philip H.

    2013-01-01

    This protocol describes the culture of human pluripotent stem cells (PSCs) under feeder-free conditions in a commercially available, chemically defined, growth medium, using Matrigel as a substrate and the enzyme solution Accutase for single-cell passaging. This system is strikingly different from traditional PSC culture, where the cells are co-cultured with feeder cells and in medium containing serum replacement. PSCs cultured in this new system have a different morphology than those cultured on feeder cells but retain their characteristic pluripotency. This feeder-free PSC culture system is conceptually similar to feeder-free systems that use mouse embryonic fibroblast (MEF)-conditioned medium (MEF-CM) and Matrigel substratum. Instead of MEF-CM, a very complex and undefined medium, this new system uses StemPro SFM, a chemically defined medium that permits enzymatic passaging with Accutase to disaggregate the colonies into single cells. Accutase passaging has been used in conjunction with Stempro in our hands for 20+ passages without detectable karyotypic abnormalities. We will also review techniques for adapting cultures previously grown on MEFs, routine passaging of the cells, and cryopreservation. PMID:21822872

  3. Oxidative Stress Promotes Doxorubicin-Induced Pgp and BCRP Expression in Colon Cancer Cells Under Hypoxic Conditions.

    PubMed

    Pinzón-Daza, Martha L; Cuellar-Saenz, Yenith; Nualart, Francisco; Ondo-Mendez, Alejandro; Del Riesgo, Lilia; Castillo-Rivera, Fabio; Garzón, Ruth

    2017-01-20

    P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) are ATP binding cassette (ABC) transporters that are overexpressed in different drug-resistant cancer cell lines. In this study, we investigated whether doxorubicin promotes Pgp and/or BCRP expression to induce drug resistance in colon cancer cells under hypoxic conditions. We analyzed HIF-1α activity via ELISA, Pgp, and BCRP expression by qRT-PCR and the relationship between doxorubicin uptake and ABC transporter expression via confocal microscopy in HT-29WT and HT-29 doxorubicin-resistant colon cancer cells (HT-29DxR). These cells were treated with doxorubicin and/or CoCl2 (chemical hypoxia), and reactive oxygen species inductors. We found that the combination of chemically induced hypoxia and doxorubicin promoted Pgp mRNA expression within 24 h in HT-29WT and HT-29DxR cells. Both doxorubicin and CoCl2 alone or in combination induced Pgp and BCRP expression, as demonstrated via confocal microscopy in each of the above two cell lines. Thus, we surmised that Pgp and BCRP expression may result from synergistic effects exerted by the combination of doxorubicin-induced ROS production and HIF-1α activity under hypoxic conditions. However, HIF-1α activity disruption via the administration of E3330, an APE-1 inhibitor, downregulated Pgp expression and increased doxorubicin delivery to HT-29 cells, where it served as a substrate for Pgp, indicating the existence of an indirect relationship between Pgp expression and doxorubicin accumulation. Thus, we concluded that Pgp and BCRP expression can be regulated via cross-talk between doxorubicin and hypoxia, promoting drug resistance in HT-29 WT, and HT-29DxR cells and that this process may be ROS dependent. J. Cell. Biochem. 9999: 1-11, 2017. © 2017 Wiley Periodicals, Inc.

  4. Characterization of Phenotypic and Transcriptional Differences in Human Pluripotent Stem Cells under 2D and 3D Culture Conditions.

    PubMed

    Kamei, Ken-Ichiro; Koyama, Yoshie; Tokunaga, Yumie; Mashimo, Yasumasa; Yoshioka, Momoko; Fockenberg, Christopher; Mosbergen, Rowland; Korn, Othmar; Wells, Christine; Chen, Yong

    2016-11-01

    Human pluripotent stem cells hold great promise for applications in drug discovery and regenerative medicine. Microfluidic technology is a promising approach for creating artificial microenvironments; however, although a proper 3D microenvironment is required to achieve robust control of cellular phenotypes, most current microfluidic devices provide only 2D cell culture and do not allow tuning of physical and chemical environmental cues simultaneously. Here, the authors report a 3D cellular microenvironment plate (3D-CEP), which consists of a microfluidic device filled with thermoresponsive poly(N-isopropylacrylamide)-β-poly(ethylene glycol) hydrogel (HG), which enables systematic tuning of both chemical and physical environmental cues as well as in situ cell monitoring. The authors show that H9 human embryonic stem cells (hESCs) and 253G1 human induced pluripotent stem cells in the HG/3D-CEP system maintain their pluripotent marker expression under HG/3D-CEP self-renewing conditions. Additionally, global gene expression analyses are used to elucidate small variations among different test environments. Interestingly, the authors find that treatment of H9 hESCs under HG/3D-CEP self-renewing conditions results in initiation of entry into the neural differentiation process by induction of PAX3 and OTX1 expression. The authors believe that this HG/3D-CEP system will serve as a versatile platform for developing targeted functional cell lines and facilitate advances in drug screening and regenerative medicine.

  5. Cells Lacking β-Actin are Genetically Reprogrammed and Maintain Conditional Migratory Capacity*

    PubMed Central

    Tondeleir, Davina; Lambrechts, Anja; Müller, Matthias; Jonckheere, Veronique; Doll, Thierry; Vandamme, Drieke; Bakkali, Karima; Waterschoot, Davy; Lemaistre, Marianne; Debeir, Olivier; Decaestecker, Christine; Hinz, Boris; Staes, An; Timmerman, Evy; Colaert, Niklaas; Gevaert, Kris; Vandekerckhove, Joël; Ampe, Christophe

    2012-01-01

    Vertebrate nonmuscle cells express two actin isoforms: cytoplasmic β- and γ-actin. Because of the presence and localized translation of β-actin at the leading edge, this isoform is generally accepted to specifically generate protrusive forces for cell migration. Recent evidence also implicates β-actin in gene regulation. Cell migration without β-actin has remained unstudied until recently and it is unclear whether other actin isoforms can compensate for this cytoplasmic function and/or for its nuclear role. Primary mouse embryonic fibroblasts lacking β-actin display compensatory expression of other actin isoforms. Consistent with this preservation of polymerization capacity, β-actin knockout cells have unchanged lamellipodial protrusion rates despite a severe migration defect. To solve this paradox we applied quantitative proteomics revealing a broad genetic reprogramming of β-actin knockout cells. This also explains why reintroducing β-actin in knockout cells does not restore the affected cell migration. Pathway analysis suggested increased Rho-ROCK signaling, consistent with observed phenotypic changes. We therefore developed and tested a model explaining the phenotypes in β-actin knockout cells based on increased Rho-ROCK signaling and increased TGFβ production resulting in increased adhesion and contractility in the knockout cells. Inhibiting ROCK or myosin restores migration of β-actin knockout cells indicating that other actins compensate for β-actin in this process. Consequently, isoactins act redundantly in providing propulsive forces for cell migration, but β-actin has a unique nuclear function, regulating expression on transcriptional and post-translational levels, thereby preventing myogenic differentiation. PMID:22448045

  6. Conditioned Medium from Placental Mesenchymal Stem Cells Reduces Oxidative Stress during the Cryopreservation of Ex Vivo Expanded Umbilical Cord Blood Cells

    PubMed Central

    Kadekar, Darshana; Rangole, Sonal; Kale, Vaijayanti; Limaye, Lalita

    2016-01-01

    Background The limited cell dose in umbilical cord blood (UCB) necessitates ex vivo expansion of UCB. Further, the effective cryopreservation of these expanded cells is important in widening their use in the clinics. During cryopreservation, cells experience oxidative stress due to the generation of reactive oxygen species (ROS). Conditioned medium from mesenchymal stem cells (MSCs-CM) has been shown to alleviate the oxidative stress during wound healing, Alzheimer’s disease and ischemic disease. This premise prompted us to investigate the influence of MSCs-CM during cryopreservation of expanded UCB cells. Methodology/Principle findings CM-was collected from cord/placental MSCs(C-MSCs-CM, P-MSC-CM). UCB CD34+cells were expanded as suspension cultures in serum free medium containing cytokines for 10 days. Cells were frozen with/without C-MSCs-CM and or P-MSCs-CM in the conventional freezing medium containing 20%FCS +10%DMSO using a programmable freezer and stored in liquid nitrogen. Upon revival, cells frozen with MSCs-CM were found to be superior to cells frozen in conventional medium in terms of viability, CD34+content and clonogenecity. Priming of revived cells for 48 hrs with MSCs-CM further improved their transplantation ability, as compared to those cultured without MSCs-CM. P-MSCs-CM radically reduced the oxidative stress in cryopreserved cells, resulting in better post thaw functionality of CD34+ cells than with C-MSCs-CM. The observed cryoprotective effect of MSCs-CM was primarily due to anti-oxidative and anti-apoptotic properties of the MSCs-CM and not because of the exosomes secreted by them. Conclusions/Significance Our data suggest that MSCs-CM can serve as a valuable additive to the freezing or the priming medium for expanded UCB cells, which would increase their clinical applicability. PMID:27780236

  7. Prediction of Low-Voltage Tetrafluoromethane Emissions Based on the Operating Conditions of an Aluminium Electrolysis Cell

    NASA Astrophysics Data System (ADS)

    Dion, Lukas; Kiss, László I.; Poncsák, Sándor; Lagacé, Charles-Luc

    2016-09-01

    Greenhouse gas (GHG) generation is inherent in the production of aluminium by a technology that uses carbon anodes. Most of those GHG are composed of CO2 produced by redox reaction that occurs in the cell. However, a significant fraction of the annual GHG production is composed of perfluorocarbons (PFC) resulting from anode effects (AE). Multiple investigations have shown that tetrafluoromethane (CF4) can be generated under low-voltage conditions in the electrolysis cells, without global anode effect. The aim of this paper is to find a quantitative relationship between monitored cell parameters and the emissions of CF4. To achieve this goal, a predictive algorithm has been developed using seven cell indicators. These indicators are based on the cell voltage, the noise level and other parameters calculated from individual anode current monitoring. The predictive algorithm is structured into three different steps. The first two steps give qualitative information while the third one quantitatively describes the expected CF4 concentration at the duct end of the electrolysis cells. Validations after each step are presented and discussed. Finally, a sensitivity analysis was performed to understand the effect of each indicator on the onset of low-voltage PFC emissions. The standard deviation of individual anode currents was found to be the dominant variable. Cell voltage, noise level, and maximum individual anode current also showed a significant correlation with the presence of CF4 in the output gas of an electrolysis cell.

  8. Lasting effect of preceding culture conditions on the susceptibility of C6 cells to peroxide-induced oxidative stress.

    PubMed

    Brenner, Sibylle; Gülden, Michael; Maser, Edmund; Seibert, Hasso

    2010-12-01

    The aim of the present study was to investigate the influence of the maintenance culture conditions on the competence of C6 rat glioma cells to cope with peroxide-induced oxidative stress. C6 cells were maintained either in Ham's nutrient mixture F-10 supplemented with 15% horse serum and 2.5% foetal bovine serum (FBS) or in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 5% FBS. The differently cultured cells were exposed under identical conditions to hydrogen peroxide (H₂O₂) and cumene hydroperoxide (CHP) in serum-free DMEM. The cells maintained in high serum Ham's F-10 medium (1) were less sensitive towards the cytotoxic action of both peroxides (EC₅₀-values: H₂O₂: 193 ± 23 μM; CHP: 94 ± 16 μM) than the cells maintained in low serum DMEM (EC₅₀-values: H₂O₂: 51 ± 10 μM; CHP: 27 ± 11 μM), (2) eliminated the peroxides (initial concentration: 100 μM) with higher rates (H₂O₂: 56 ± 5.5 vs. 32 ± 2.7, CHP: 32 ± 6 vs. 3.4 ± 0.6 nmol/min mg protein), (3) contained more glutathione (30 ± 2.5 vs. 14 ± 1.1 nmol/mg protein) and (4) owned a higher glutathione peroxidase activity (28 ± 3.4 vs. 9.5 ± 0.8 mU/mg protein). Glutathione reductase and catalase activities were not affected. These results demonstrate that the preceding culture conditions have a lasting effect on the susceptibility of cultured cells to oxidative stressors like peroxides. As cause for these differences a dissimilar supply of the cells with serum born antioxidants like selenium and α-tocopherol is discussed.

  9. Hydrogen sulfide poisoning in solid oxide fuel cells under accelerated testing conditions

    NASA Astrophysics Data System (ADS)

    Li, Ting Shuai; Wang, Wei Guo; Chen, Tao; Miao, He; Xu, Cheng

    This study investigates the 0.2% hydrogen sulfide poisoning of Ni/YSZ anode-supported solid oxide fuel cells (SOFCs). The deterioration degrees and recovery extents of the cell current density, cell voltage and operation temperature are monitored. The results of impedance spectroscopy analysis show that hydrogen sulfide poisoning behavior may affect oxygen ion migration and gas diffusion and conversion on the anode side. Microstructural inspection reveals sulfur or sulfide formed on the anode-active area, which accounts for the immediate and severe cell power drop upon the injection of H 2S. The nickel sulfide in the anodic functional layer cannot be completely removed after long-term regeneration and thus may be a key factor in the permanent degradation of the cell.

  10. Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

    PubMed Central

    Ganguly, Anutosh; Zhang, Hong; Sharma, Ritu; Parsons, Sean; Patel, Kamala D.

    2012-01-01

    Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells. One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-α results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8. The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow

  11. Hypoxic culture conditions induce increased metabolic rate and collagen gene expression in ACL-derived cells.

    PubMed

    Kowalski, Tomasz J; Leong, Natalie L; Dar, Ayelet; Wu, Ling; Kabir, Nima; Khan, Adam Z; Eliasberg, Claire D; Pedron, Andrew; Karayan, Ashant; Lee, Siyoung; Di Pauli von Treuheim, Theodor; Jiacheng, Jin; Wu, Ben M; Evseenko, Denis; McAllister, David R; Petrigliano, Frank A

    2016-06-01

    There has been substantial effort directed toward the application of bone marrow and adipose-derived mesenchymal stromal cells (MSCs) in the regeneration of musculoskeletal tissue. Recently, resident tissue-specific stem cells have been described in a variety of mesenchymal structures including ligament, tendon, muscle, cartilage, and bone. In the current study, we systematically characterize three novel anterior cruciate ligament (ACL)-derived cell populations with the potential for ligament regeneration: ligament-forming fibroblasts (LFF: CD146(neg) , CD34(neg) CD44(pos) , CD31(neg) , CD45(neg) ), ligament perivascular cells (LPC: CD146(pos) CD34(neg) CD44(pos) , CD31(neg) , CD45(neg) ) and ligament interstitial cells (LIC: CD34(pos) CD146(neg) , CD44(pos) , CD31(neg) , CD45(neg) )-and describe their proliferative and differentiation potential, collagen gene expression and metabolism in both normoxic and hypoxic environments, and their trophic potential in vitro. All three groups of cells (LIC, LPC, and LFF) isolated from adult human ACL exhibited progenitor cell characteristics with regard to proliferation and differentiation potential in vitro. Culture in low oxygen tension enhanced the collagen I and III gene expression in LICs (by 2.8- and 3.3-fold, respectively) and LFFs (by 3- and 3.5-fold, respectively) and increased oxygen consumption rate and extracellular acidification rate in LICs (by 4- and 3.5-fold, respectively), LFFs (by 5.5- and 3-fold, respectively), LPCs (by 10- and 4.5-fold, respectively) as compared to normal oxygen concentration. In summary, this study demonstrates for the first time the presence of three novel progenitor cell populations in the adult ACL that demonstrate robust proliferative and matrix synthetic capacity; these cells may play a role in local ligament regeneration, and consequently represent a potential cell source for ligament engineering applications. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc

  12. Chondrogenic differentiation of human adipose-derived stem cells in polyglycolic acid mesh scaffolds under dynamic culture conditions.

    PubMed

    Mahmoudifar, Nastaran; Doran, Pauline M

    2010-05-01

    Chondrogenic differentiation of human adult adipose-derived stem cells was studied in vitro for the development of engineered cartilage tissue. Cells cultured under dynamic conditions in polyglycolic acid (PGA) scaffolds produced substantially higher glycosaminoglycan (GAG) and total collagen levels than cells in pellet cultures. This result reflects the importance of cell attachment and cell-scaffold interactions in stem cell differentiation and chondrogenesis. Although gene expression levels for both aggrecan and collagen type II were up-regulated significantly in PGA cultures treated with transforming growth factor beta1 (TGF-beta1), synthesis of GAG but not collagen type II was enhanced in tissue constructs when TGF-beta1 was added to the medium. Bone morphogenetic protein-6 (BMP-6) in the presence of TGF-beta1 was effective in improving GAG and total collagen production when the cells were pre-treated with fibroblast growth factor-2 (FGF-2) prior to scaffold seeding. Extending the culture duration from 2 to 5 weeks did not improve cartilage development in PGA scaffolds; loss of cells from the constructs suggested that the rate of scaffold degradation exceeded the rate of replacement by ECM during the 5-week period. Stem cells in PGA scaffolds were cultured in perfusion-type recirculation bioreactors operated with periodic medium flow reversal. The highest levels of GAG and collagen type II accumulation were achieved in the bioreactor cultures after the seeding cell density was increased from 2x10(7) to 4x10(7) cells per scaffold.

  13. A trial of plerixafor adjunctive therapy in allogeneic hematopoietic cell transplantation with minimal conditioning for severe combined immunodeficiency.

    PubMed

    Dvorak, Christopher C; Horn, Biljana N; Puck, Jennifer M; Czechowicz, Agnieszka; Shizuru, Judy A; Ko, Rose M; Cowan, Morton J

    2014-09-01

    For infants with SCID, the ideal conditioning regimen before allogeneic HCT would omit cytotoxic chemotherapy to minimize short- and long-term complications. We performed a prospective pilot trial with G-CSF plus plerixafor given to the host to mobilize HSC from their niches. We enrolled six patients who received CD34-selected haploidentical cells and one who received T-replete matched unrelated BM. All patients receiving G-CSF and plerixafor had generally poor CD34(+) cell and Lin(-) CD34(+) CD38(-) CD90(+) CD45RA(-) HSC mobilization, and developed donor T cells, but no donor myeloid or B-cell engraftment. Although well tolerated, G-CSF plus plerixafor alone failed to overcome physical barriers to donor engraftment.

  14. In Situ Irradiation and Measurement of Triple Junction Solar Cells at Low Intensity, Low Temperature (LILT) Conditions

    NASA Technical Reports Server (NTRS)

    Harris, R.D.; Imaizumi, M.; Walters, R.J.; Lorentzen, J.R.; Messenger, S.R.; Tischler, J.G.; Ohshima, T.; Sato, S.; Sharps, P.R.; Fatemi, N.S.

    2008-01-01

    The performance of triple junction InGaP/(In)GaAs/Ge space solar cells was studied following high energy electron irradiation at low temperature. Cell characterization was carried out in situ at the irradiation temperature while using low intensity illumination, and, as such, these conditions reflect those found for deep space, solar powered missions that are far from the sun. Cell characterization consisted of I-V measurements and quantum efficiency measurements. The low temperature irradiations caused substantial degradation that differs in some ways from that seen after room temperature irradiations. The short circuit current degrades more at low temperature while the open circuit voltage degrades more at room temperature. A room temperature anneal after the low temperature irradiation produced a substantial recovery in the degradation. Following irradiation at both temperatures and an extended room temperature anneal, quantum efficiency measurement suggests that the bulk of the remaining damage is in the (In)GaAs sub-cell

  15. Combining characteristic forms of boundary conditions and conservation equations at boundaries of cell-centered Euler-flow calculations

    NASA Astrophysics Data System (ADS)

    Boerstoel, J. W.

    1987-04-01

    A numerical method to obtain the additional equations in Euler-flow calculations based on cell-centered schemes when the number of equations required to determine the flow-state evaluation at grid points half a mesh outside the flow domain exceeds the number of boundary-condition equations provided by characteristic theory, is presented. A layer of auxiliary cells on flow boundaries is introduced, and semidiscrete conservation equations for these cells are defined. The time variations of the state in these auxiliary cells at the boundary are transformed into characteristic form, and time variations of characteristic variables corresponding to incoming information from the boundary into the flow are replaced by boundary conditions for these time variations. The boundary equations so obtained are mapped back into a form with primitive variables, and numerically integrated in time. The characteristic boundary conditions are first-order differential equations for time variations at boundary points of characteristic variables. These equations may be chosen to express that given functions of the flow state on the boundary should asymptotically tend with time to prescribed steady-state values.

  16. A thermodynamic approach for selecting operating conditions in the design of reversible solid oxide cell energy systems

    NASA Astrophysics Data System (ADS)

    Wendel, Christopher H.; Kazempoor, Pejman; Braun, Robert J.

    2016-01-01

    Reversible solid oxide cell (ReSOC) systems are being increasingly considered for electrical energy storage, although much work remains before they can be realized, including cell materials development and system design optimization. These systems store electricity by generating a synthetic fuel in electrolysis mode and subsequently recover electricity by electrochemically oxidizing the stored fuel in fuel cell mode. System thermal management is improved by promoting methane synthesis internal to the ReSOC stack. Within this strategy, the cell-stack operating conditions are highly impactful on system performance and optimizing these parameters to suit both operating modes is critical to achieving high roundtrip efficiency. Preliminary analysis shows the thermoneutral voltage to be a useful parameter for analyzing ReSOC systems and the focus of this study is to quantitatively examine how it is affected by ReSOC operating conditions. The results reveal that the thermoneutral voltage is generally reduced by increased pressure, and reductions in temperature, fuel utilization, and hydrogen-to-carbon ra