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Sample records for cell-derived neural populations

  1. Niche-dependent development of functional neuronal networks from embryonic stem cell-derived neural populations

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    Siebler Mario

    2009-08-01

    Full Text Available Abstract Background The present work was performed to investigate the ability of two different embryonic stem (ES cell-derived neural precursor populations to generate functional neuronal networks in vitro. The first ES cell-derived neural precursor population was cultivated as free-floating neural aggregates which are known to form a developmental niche comprising different types of neural cells, including neural precursor cells (NPCs, progenitor cells and even further matured cells. This niche provides by itself a variety of different growth factors and extracellular matrix proteins that influence the proliferation and differentiation of neural precursor and progenitor cells. The second population was cultivated adherently in monolayer cultures to control most stringently the extracellular environment. This population comprises highly homogeneous NPCs which are supposed to represent an attractive way to provide well-defined neuronal progeny. However, the ability of these different ES cell-derived immature neural cell populations to generate functional neuronal networks has not been assessed so far. Results While both precursor populations were shown to differentiate into sufficient quantities of mature NeuN+ neurons that also express GABA or vesicular-glutamate-transporter-2 (vGlut2, only aggregate-derived neuronal populations exhibited a synchronously oscillating network activity 2–4 weeks after initiating the differentiation as detected by the microelectrode array technology. Neurons derived from homogeneous NPCs within monolayer cultures did merely show uncorrelated spiking activity even when differentiated for up to 12 weeks. We demonstrated that these neurons exhibited sparsely ramified neurites and an embryonic vGlut2 distribution suggesting an inhibited terminal neuronal maturation. In comparison, neurons derived from heterogeneous populations within neural aggregates appeared as fully mature with a dense neurite network and punctuated

  2. Neural stem cell-derived exosomes mediate viral entry

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    Sims B

    2014-10-01

    Full Text Available Brian Sims,1,2,* Linlin Gu,3,* Alexandre Krendelchtchikov,3 Qiana L Matthews3,4 1Division of Neonatology, Department of Pediatrics, 2Department of Cell, Developmental, and Integrative Biology, 3Division of Infectious Diseases, Department of Medicine, 4Center for AIDS Research, University of Alabama at Birmingham, Birmingham, AL, USA *These authors contributed equally to this work Background: Viruses enter host cells through interactions of viral ligands with cellular receptors. Viruses can also enter cells in a receptor-independent fashion. Mechanisms regarding the receptor-independent viral entry into cells have not been fully elucidated. Exosomal trafficking between cells may offer a mechanism by which viruses can enter cells.Methods: To investigate the role of exosomes on cellular viral entry, we employed neural stem cell-derived exosomes and adenovirus type 5 (Ad5 for the proof-of-principle study. Results: Exosomes significantly enhanced Ad5 entry in Coxsackie virus and adenovirus receptor (CAR-deficient cells, in which Ad5 only had very limited entry. The exosomes were shown to contain T-cell immunoglobulin mucin protein 4 (TIM-4, which binds phosphatidylserine. Treatment with anti-TIM-4 antibody significantly blocked the exosome-mediated Ad5 entry.Conclusion: Neural stem cell-derived exosomes mediated significant cellular entry of Ad5 in a receptor-independent fashion. This mediation may be hampered by an antibody specifically targeting TIM-4 on exosomes. This set of results will benefit further elucidation of virus/exosome pathways, which would contribute to reducing natural viral infection by developing therapeutic agents or vaccines. Keywords: neural stem cell-derived exosomes, adenovirus type 5, TIM-4, viral entry, phospholipids

  3. Purification of human induced pluripotent stem cell-derived neural precursors using magnetic activated cell sorting.

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    Rodrigues, Gonçalo M C; Fernandes, Tiago G; Rodrigues, Carlos A V; Cabral, Joaquim M S; Diogo, Maria Margarida

    2015-01-01

    Neural precursor (NP) cells derived from human induced pluripotent stem cells (hiPSCs), and their neuronal progeny, will play an important role in disease modeling, drug screening tests, central nervous system development studies, and may even become valuable for regenerative medicine treatments. Nonetheless, it is challenging to obtain homogeneous and synchronously differentiated NP populations from hiPSCs, and after neural commitment many pluripotent stem cells remain in the differentiated cultures. Here, we describe an efficient and simple protocol to differentiate hiPSC-derived NPs in 12 days, and we include a final purification stage where Tra-1-60+ pluripotent stem cells (PSCs) are removed using magnetic activated cell sorting (MACS), leaving the NP population nearly free of PSCs.

  4. Functional integration of grafted neural stem cell-derived dopaminergic neurons monitored by optogenetics in an in vitro Parkinson model

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    Tønnesen, Jan; Parish, Clare L; Sørensen, Andreas T

    2011-01-01

    Intrastriatal grafts of stem cell-derived dopamine (DA) neurons induce behavioral recovery in animal models of Parkinson's disease (PD), but how they functionally integrate in host neural circuitries is poorly understood. Here, Wnt5a-overexpressing neural stem cells derived from embryonic ventral...... of post-synaptic currents, and functional expression of DA D₂ autoreceptors. These properties resembled those recorded from identical cells in acute slices of intrastriatal grafts in the 6-hydroxy-DA-induced mouse PD model and from DA neurons in intact substantia nigra. Optogenetic activation...... using optogenetics that ectopically grafted stem cell-derived DA neurons become functionally integrated in the DA-denervated striatum. Further optogenetic dissection of the synaptic wiring between grafted and host neurons will be crucial to clarify the cellular and synaptic mechanisms underlying...

  5. Production of hemizygous and homozygous embryonic stem cell-derived neural progenitor cells from the transgenic alszheimer göttingen minipis

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    Hall, Vanessa Jane; Jacobsen, J.; Gunnarsson, A.

    2011-01-01

    Production of hemizygous and homozygous embryonic stem cell-derived neural progenitor cells from the transgenic alszheimer göttingen minipis......Production of hemizygous and homozygous embryonic stem cell-derived neural progenitor cells from the transgenic alszheimer göttingen minipis...

  6. The postischemic environment differentially impacts teratoma or tumor formation after transplantation of human embryonic stem cell-derived neural progenitors

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    Seminatore, Christine; Polentes, Jerome; Ellman, Ditte

    2010-01-01

    Risk of tumorigenesis is a major obstacle to human embryonic and induced pluripotent stem cell therapy. Likely linked to the stage of differentiation of the cells at the time of implantation, formation of teratoma/tumors can also be influenced by factors released by the host tissue. We have...... analyzed the relative effects of the stage of differentiation and the postischemic environment on the formation of adverse structures by transplanted human embryonic stem cell-derived neural progenitors....

  7. Functional integration of grafted neural stem cell-derived dopaminergic neurons monitored by optogenetics in an in vitro Parkinson model.

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    Jan Tønnesen

    Full Text Available Intrastriatal grafts of stem cell-derived dopamine (DA neurons induce behavioral recovery in animal models of Parkinson's disease (PD, but how they functionally integrate in host neural circuitries is poorly understood. Here, Wnt5a-overexpressing neural stem cells derived from embryonic ventral mesencephalon of tyrosine hydroxylase-GFP transgenic mice were expanded as neurospheres and transplanted into organotypic cultures of wild type mouse striatum. Differentiated GFP-labeled DA neurons in the grafts exhibited mature neuronal properties, including spontaneous firing of action potentials, presence of post-synaptic currents, and functional expression of DA D₂ autoreceptors. These properties resembled those recorded from identical cells in acute slices of intrastriatal grafts in the 6-hydroxy-DA-induced mouse PD model and from DA neurons in intact substantia nigra. Optogenetic activation or inhibition of grafted cells and host neurons using channelrhodopsin-2 (ChR2 and halorhodopsin (NpHR, respectively, revealed complex, bi-directional synaptic interactions between grafted cells and host neurons and extensive synaptic connectivity within the graft. Our data demonstrate for the first time using optogenetics that ectopically grafted stem cell-derived DA neurons become functionally integrated in the DA-denervated striatum. Further optogenetic dissection of the synaptic wiring between grafted and host neurons will be crucial to clarify the cellular and synaptic mechanisms underlying behavioral recovery as well as adverse effects following stem cell-based DA cell replacement strategies in PD.

  8. Human induced pluripotent stem cell-derived models to investigate human cytomegalovirus infection in neural cells.

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    Leonardo D'Aiuto

    Full Text Available Human cytomegalovirus (HCMV infection is one of the leading prenatal causes of congenital mental retardation and deformities world-wide. Access to cultured human neuronal lineages, necessary to understand the species specific pathogenic effects of HCMV, has been limited by difficulties in sustaining primary human neuronal cultures. Human induced pluripotent stem (iPS cells now provide an opportunity for such research. We derived iPS cells from human adult fibroblasts and induced neural lineages to investigate their susceptibility to infection with HCMV strain Ad169. Analysis of iPS cells, iPS-derived neural stem cells (NSCs, neural progenitor cells (NPCs and neurons suggests that (i iPS cells are not permissive to HCMV infection, i.e., they do not permit a full viral replication cycle; (ii Neural stem cells have impaired differentiation when infected by HCMV; (iii NPCs are fully permissive for HCMV infection; altered expression of genes related to neural metabolism or neuronal differentiation is also observed; (iv most iPS-derived neurons are not permissive to HCMV infection; and (v infected neurons have impaired calcium influx in response to glutamate.

  9. [Investigation of neural stem cell-derived donor contribution in the inner ear following blastocyst injection].

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    Volkenstein, S; Brors, D; Hansen, S; Mlynski, R; Dinger, T C; Müller, A M; Dazert, S

    2008-03-01

    Utilising the enormous proliferation and multi-lineage differentiation potentials of somatic stem cells represents a possible therapeutical strategy for diseases of non-regenerative tissues like the inner ear. In the current study, the possibility of murine neural stem cells to contribute to the developing inner ear following blastocyst injection was investigated. Fetal brain-derived neural stem cells from the embryonic day 14 cortex of male mice were isolated and expanded for four weeks in neurobasal media supplemented with bFGF and EGF. Neural stem cells of male animals were harvested, injected into blastocysts and the blastocysts were transferred into pseudo-pregnant foster animals. Each blastocyst was injected with 5-15 microspheres growing from single cell suspension from neurospheres dissociated the day before. The resulting mice were investigated six months POST PARTUM for the presence of donor cells. Brainstem evoked response audiometry (BERA) was performed in six animals. To visualize donor cells Lac-Z staining was performed on sliced cochleas of two animals. In addition, the cochleas of four female animals were isolated and genomic DNA of the entire cochlea was analyzed for donor contribution by Y-chromosome-specific PCR. All animals had normal thresholds in brainstem evoked response audiometry. The male-specific PCR product indicating the presence of male donor cells were detected in the cochleas of three of the four female animals investigated. In two animals, male donor cells were detected unilateral, in one animal bilateral. The results suggest that descendants of neural stem cells are detectable in the inner ear after injection into blastocysts and possess the ability to integrate into the developing inner ear without obvious loss in hearing function.

  10. Induced Pluripotent Stem Cell-Derived Neural Cells Survive and Mature in the Nonhuman Primate Brain

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    Marina E. Emborg

    2013-03-01

    Full Text Available The generation of induced pluripotent stem cells (iPSCs opens up the possibility for personalized cell therapy. Here, we show that transplanted autologous rhesus monkey iPSC-derived neural progenitors survive for up to 6 months and differentiate into neurons, astrocytes, and myelinating oligodendrocytes in the brains of MPTP-induced hemiparkinsonian rhesus monkeys with a minimal presence of inflammatory cells and reactive glia. This finding represents a significant step toward personalized regenerative therapies.

  11. Induced pluripotent stem cell-derived neural cells survive and mature in the nonhuman primate brain.

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    Emborg, Marina E; Liu, Yan; Xi, Jiajie; Zhang, Xiaoqing; Yin, Yingnan; Lu, Jianfeng; Joers, Valerie; Swanson, Christine; Holden, James E; Zhang, Su-Chun

    2013-03-28

    The generation of induced pluripotent stem cells (iPSCs) opens up the possibility for personalized cell therapy. Here, we show that transplanted autologous rhesus monkey iPSC-derived neural progenitors survive for up to 6 months and differentiate into neurons, astrocytes, and myelinating oligodendrocytes in the brains of MPTP-induced hemiparkinsonian rhesus monkeys with a minimal presence of inflammatory cells and reactive glia. This finding represents a significant step toward personalized regenerative therapies. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  12. In vitro generation of three-dimensional substrate-adherent embryonic stem cell-derived neural aggregates for application in animal models of neurological disorders.

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    Hargus, Gunnar; Cui, Yi-Fang; Dihné, Marcel; Bernreuther, Christian; Schachner, Melitta

    2012-05-01

    In vitro-differentiated embryonic stem (ES) cells comprise a useful source for cell replacement therapy, but the efficiency and safety of a translational approach are highly dependent on optimized protocols for directed differentiation of ES cells into the desired cell types in vitro. Furthermore, the transplantation of three-dimensional ES cell-derived structures instead of a single-cell suspension may improve graft survival and function by providing a beneficial microenvironment for implanted cells. To this end, we have developed a new method to efficiently differentiate mouse ES cells into neural aggregates that consist predominantly (>90%) of postmitotic neurons, neural progenitor cells, and radial glia-like cells. When transplanted into the excitotoxically lesioned striatum of adult mice, these substrate-adherent embryonic stem cell-derived neural aggregates (SENAs) showed significant advantages over transplanted single-cell suspensions of ES cell-derived neural cells, including improved survival of GABAergic neurons, increased cell migration, and significantly decreased risk of teratoma formation. Furthermore, SENAs mediated functional improvement after transplantation into animal models of Parkinson's disease and spinal cord injury. This unit describes in detail how SENAs are efficiently derived from mouse ES cells in vitro and how SENAs are isolated for transplantation. Furthermore, methods are presented for successful implantation of SENAs into animal models of Huntington's disease, Parkinson's disease, and spinal cord injury to study the effects of stem cell-derived neural aggregates in a disease context in vivo.

  13. Hypoxia Epigenetically Confers Astrocytic Differentiation Potential on Human Pluripotent Cell-Derived Neural Precursor Cells

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    Tetsuro Yasui

    2017-06-01

    Full Text Available Human neural precursor cells (hNPCs derived from pluripotent stem cells display a high propensity for neuronal differentiation, but they require long-term culturing to differentiate efficiently into astrocytes. The mechanisms underlying this biased fate specification of hNPCs remain elusive. Here, we show that hypoxia confers astrocytic differentiation potential on hNPCs through epigenetic gene regulation, and that this was achieved by cooperation between hypoxia-inducible factor 1α and Notch signaling, accompanied by a reduction of DNA methylation level in the promoter region of a typical astrocyte-specific gene, Glial fibrillary acidic protein. Furthermore, we found that this hypoxic culture condition could be applied to rapid generation of astrocytes from Rett syndrome patient-derived hNPCs, and that these astrocytes impaired neuronal development. Thus, our findings shed further light on the molecular mechanisms regulating hNPC differentiation and provide attractive tools for the development of therapeutic strategies for treating astrocyte-mediated neurological disorders.

  14. Intraocular Injection of ES Cell-Derived Neural Progenitors Improve Visual Function in Retinal Ganglion Cell-Depleted Mouse Models

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    Mundackal S. Divya

    2017-09-01

    Full Text Available Retinal ganglion cell (RGC transplantation is a promising strategy to restore visual function resulting from irreversible RGC degeneration occurring in glaucoma or inherited optic neuropathies. We previously demonstrated FGF2 induced differentiation of mouse embryonic stem cells (ESC to RGC lineage, capable of retinal ganglion cell layer (GCL integration upon transplantation. Here, we evaluated possible improvement of visual function by transplantation of ES cell derived neural progenitors in RGC depleted glaucoma mice models. ESC derived neural progenitors (ES-NP were transplanted into N-Methyl-D-Aspartate (NMDA injected, RGC-ablated mouse models and a pre-clinical glaucoma mouse model (DBA/2J having sustained higher intra ocular pressure (IOP. Visual acuity and functional integration was evaluated by behavioral experiments and immunohistochemistry, respectively. GFP-expressing ES-NPs transplanted in NMDA-injected RGC-depleted mice differentiated into RGC lineage and possibly integrating into GCL. An improvement in visual acuity was observed after 2 months of transplantation, when compared to the pre-transplantation values. Expression of c-Fos in the transplanted cells, upon light induction, further suggests functional integration into the host retinal circuitry. However, the transplanted cells did not send axonal projections into optic nerve. Transplantation experiments in DBA/2J mouse showed no significant improvement in visual functions, possibly due to both host and transplanted retinal cell death which could be due to an inherent high IOP. We showed that, ES NPs transplanted into the retina of RGC-ablated mouse models could survive, differentiate to RGC lineage, and possibly integrate into GCL to improve visual function. However, for the survival of transplanted cells in glaucoma, strategies to control the IOP are warranted.

  15. A Human Neural Crest Stem Cell-Derived Dopaminergic Neuronal Model Recapitulates Biochemical Abnormalities in GBA1 Mutation Carriers

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    Shi-Yu Yang

    2017-03-01

    Full Text Available Numerically the most important risk factor for the development of Parkinson's disease (PD is the presence of mutations in the glucocerebrosidase GBA1 gene. In vitro and in vivo studies show that GBA1 mutations reduce glucocerebrosidase (GCase activity and are associated with increased α-synuclein levels, reflecting similar changes seen in idiopathic PD brain. We have developed a neural crest stem cell-derived dopaminergic neuronal model that recapitulates biochemical abnormalities in GBA1 mutation-associated PD. Cells showed reduced GCase protein and activity, impaired macroautophagy, and increased α-synuclein levels. Advantages of this approach include easy access to stem cells, no requirement to reprogram, and retention of the intact host genome. Treatment with a GCase chaperone increased GCase protein levels and activity, rescued the autophagic defects, and decreased α-synuclein levels. These results provide the basis for further investigation of GCase chaperones or similar drugs to slow the progression of PD.

  16. Intrinsically active and pacemaker neurons in pluripotent stem cell-derived neuronal populations.

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    Illes, Sebastian; Jakab, Martin; Beyer, Felix; Gelfert, Renate; Couillard-Despres, Sébastien; Schnitzler, Alfons; Ritter, Markus; Aigner, Ludwig

    2014-03-11

    Neurons generated from pluripotent stem cells (PSCs) self-organize into functional neuronal assemblies in vitro, generating synchronous network activities. Intriguingly, PSC-derived neuronal assemblies develop spontaneous activities that are independent of external stimulation, suggesting the presence of thus far undetected intrinsically active neurons (IANs). Here, by using mouse embryonic stem cells, we provide evidence for the existence of IANs in PSC-neuronal networks based on extracellular multielectrode array and intracellular patch-clamp recordings. IANs remain active after pharmacological inhibition of fast synaptic communication and possess intrinsic mechanisms required for autonomous neuronal activity. PSC-derived IANs are functionally integrated in PSC-neuronal populations, contribute to synchronous network bursting, and exhibit pacemaker properties. The intrinsic activity and pacemaker properties of the neuronal subpopulation identified herein may be particularly relevant for interventions involving transplantation of neural tissues. IANs may be a key element in the regulation of the functional activity of grafted as well as preexisting host neuronal networks.

  17. A chemically defined substrate for the expansion and neuronal differentiation of human pluripotent stem cell-derived neural progenitor cells

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    Yihuan Tsai

    2015-07-01

    Full Text Available Due to the limitation of current pharmacological therapeutic strategies, stem cell therapies have emerged as a viable option for treating many incurable neurological disorders. Specifically, human pluripotent stem cell (hPSC-derived neural progenitor cells (hNPCs, a multipotent cell population that is capable of near indefinite expansion and subsequent differentiation into the various cell types that comprise the central nervous system (CNS, could provide an unlimited source of cells for such cell-based therapies. However the clinical application of these cells will require (i defined, xeno-free conditions for their expansion and neuronal differentiation and (ii scalable culture systems that enable their expansion and neuronal differentiation in numbers sufficient for regenerative medicine and drug screening purposes. Current extracellular matrix protein (ECMP-based substrates for the culture of hNPCs are expensive, difficult to isolate, subject to batch-to-batch variations, and, therefore, unsuitable for clinical application of hNPCs. Using a high-throughput array-based screening approach, we identified a synthetic polymer, poly(4-vinyl phenol (P4VP, that supported the long-term proliferation and self-renewal of hNPCs. The hNPCs cultured on P4VP maintained their characteristic morphology, expressed high levels of markers of multipotency, and retained their ability to differentiate into neurons. Such chemically defined substrates will eliminate critical roadblocks for the utilization of hNPCs for human neural regenerative repair, disease modeling, and drug discovery.

  18. Stromal cell-derived factor-1 alpha (SDF-1 alpha) improves neural recovery after spinal cord contusion in rats

    NARCIS (Netherlands)

    Zendedel, A.; Nobakht, M.; Bakhtiyari, M.; Beyer, C.; Kipp, M.; Baazm, M.; Joghataie, M.T.

    2012-01-01

    Stromal cell-derived factor-1 alpha (SDF-1α) is an important cytokine, implicated in the control of stem cell trafficking and bone marrow-derived stem cell mobilization. Generally, SDF-1α regulates multiple physiological processes such as embryonic development and organ homeostasis. There is also

  19. A chemically defined substrate for the expansion and neuronal differentiation of human pluripotent stem cell-derived neural progenitor cells.

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    Tsai, Yihuan; Cutts, Josh; Kimura, Azuma; Varun, Divya; Brafman, David A

    2015-07-01

    Due to the limitation of current pharmacological therapeutic strategies, stem cell therapies have emerged as a viable option for treating many incurable neurological disorders. Specifically, human pluripotent stem cell (hPSC)-derived neural progenitor cells (hNPCs), a multipotent cell population that is capable of near indefinite expansion and subsequent differentiation into the various cell types that comprise the central nervous system (CNS), could provide an unlimited source of cells for such cell-based therapies. However the clinical application of these cells will require (i) defined, xeno-free conditions for their expansion and neuronal differentiation and (ii) scalable culture systems that enable their expansion and neuronal differentiation in numbers sufficient for regenerative medicine and drug screening purposes. Current extracellular matrix protein (ECMP)-based substrates for the culture of hNPCs are expensive, difficult to isolate, subject to batch-to-batch variations, and, therefore, unsuitable for clinical application of hNPCs. Using a high-throughput array-based screening approach, we identified a synthetic polymer, poly(4-vinyl phenol) (P4VP), that supported the long-term proliferation and self-renewal of hNPCs. The hNPCs cultured on P4VP maintained their characteristic morphology, expressed high levels of markers of multipotency, and retained their ability to differentiate into neurons. Such chemically defined substrates will eliminate critical roadblocks for the utilization of hNPCs for human neural regenerative repair, disease modeling, and drug discovery. Copyright © 2015. Published by Elsevier B.V.

  20. Vitamin E-Mediated Modulation of Glutamate Receptor Expression in an Oxidative Stress Model of Neural Cells Derived from Embryonic Stem Cell Cultures

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    Afifah Abd Jalil

    2017-01-01

    Full Text Available Glutamate is the primary excitatory neurotransmitter in the central nervous system. Excessive concentrations of glutamate in the brain can be excitotoxic and cause oxidative stress, which is associated with Alzheimer’s disease. In the present study, the effects of vitamin E in the form of tocotrienol-rich fraction (TRF and alpha-tocopherol (α-TCP in modulating the glutamate receptor and neuron injury markers in an in vitro model of oxidative stress in neural-derived embryonic stem (ES cell cultures were elucidated. A transgenic mouse ES cell line (46C was differentiated into a neural lineage in vitro via induction with retinoic acid. These cells were then subjected to oxidative stress with a significantly high concentration of glutamate. Measurement of reactive oxygen species (ROS was performed after inducing glutamate excitotoxicity, and recovery from this toxicity in response to vitamin E was determined. The gene expression levels of glutamate receptors and neuron-specific enolase were elucidated using real-time PCR. The results reveal that neural cells derived from 46C cells and subjected to oxidative stress exhibit downregulation of NMDA, kainate receptor, and NSE after posttreatment with different concentrations of TRF and α-TCP, a sign of neurorecovery. Treatment of either TRF or α-TCP reduced the levels of ROS in neural cells subjected to glutamate-induced oxidative stress; these results indicated that vitamin E is a potent antioxidant.

  1. Neurite extension and neuronal differentiation of human induced pluripotent stem cell derived neural stem cells on polyethylene glycol hydrogels containing a continuous Young's Modulus gradient.

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    Mosley, Matthew C; Lim, Hyun Ju; Chen, Jing; Yang, Yueh-Hsun; Li, Shenglan; Liu, Ying; Smith Callahan, Laura A

    2017-03-01

    Mechanotransduction in neural cells involves multiple signaling pathways that are not fully understood. Differences in lineage and maturation state are suggested causes for conflicting reports on neural cell mechanosensitivity. To optimize matrices for use in stem cell therapy treatments transplanting human induced pluripotent stem cell derived neural stem cells (hNSC) into lesions after spinal cord injury, the effects of Young's Modulus changes on hNSC behavior must be understood. The present study utilizes polyethylene glycol hydrogels containing a continuous gradient in Young's modulus to examine changes in the Young's Modulus of the culture substrate on hNSC neurite extension and neural differentiation. Changes in the Young's Modulus of the polyethylene glycol hydrogels was found to affect neurite extension and cellular organization on the matrices. hNSC cultured on 907 Pa hydrogels were found to extend longer neurites than hNSC cultured on other tested Young's Moduli hydrogels. The gene expression of β tubulin III and microtubule-associated protein 2 in hNSC was affected by changes in the Young's Modulus of the hydrogel. The combinatory method approach used in the present study demonstrates that hNSC are mechanosensitive and the matrix Young's Modulus should be a design consideration for hNSC transplant applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 824-833, 2017. © 2016 Wiley Periodicals, Inc.

  2. GABA and Gap Junctions in the Development of Synchronized Activity in Human Pluripotent Stem Cell-Derived Neural Networks

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    Meeri Eeva-Liisa Mäkinen

    2018-03-01

    Full Text Available The electrical activity of the brain arises from single neurons communicating with each other. However, how single neurons interact during early development to give rise to neural network activity remains poorly understood. We studied the emergence of synchronous neural activity in human pluripotent stem cell (hPSC-derived neural networks simultaneously on a single-neuron level and network level. The contribution of gamma-aminobutyric acid (GABA and gap junctions to the development of synchronous activity in hPSC-derived neural networks was studied with GABA agonist and antagonist and by blocking gap junctional communication, respectively. We characterized the dynamics of the network-wide synchrony in hPSC-derived neural networks with high spatial resolution (calcium imaging and temporal resolution microelectrode array (MEA. We found that the emergence of synchrony correlates with a decrease in very strong GABA excitation. However, the synchronous network was found to consist of a heterogeneous mixture of synchronously active cells with variable responses to GABA, GABA agonists and gap junction blockers. Furthermore, we show how single-cell distributions give rise to the network effect of GABA, GABA agonists and gap junction blockers. Finally, based on our observations, we suggest that the earliest form of synchronous neuronal activity depends on gap junctions and a decrease in GABA induced depolarization but not on GABAA mediated signaling.

  3. GABA and Gap Junctions in the Development of Synchronized Activity in Human Pluripotent Stem Cell-Derived Neural Networks

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    Mäkinen, Meeri Eeva-Liisa; Ylä-Outinen, Laura; Narkilahti, Susanna

    2018-01-01

    The electrical activity of the brain arises from single neurons communicating with each other. However, how single neurons interact during early development to give rise to neural network activity remains poorly understood. We studied the emergence of synchronous neural activity in human pluripotent stem cell (hPSC)-derived neural networks simultaneously on a single-neuron level and network level. The contribution of gamma-aminobutyric acid (GABA) and gap junctions to the development of synchronous activity in hPSC-derived neural networks was studied with GABA agonist and antagonist and by blocking gap junctional communication, respectively. We characterized the dynamics of the network-wide synchrony in hPSC-derived neural networks with high spatial resolution (calcium imaging) and temporal resolution microelectrode array (MEA). We found that the emergence of synchrony correlates with a decrease in very strong GABA excitation. However, the synchronous network was found to consist of a heterogeneous mixture of synchronously active cells with variable responses to GABA, GABA agonists and gap junction blockers. Furthermore, we show how single-cell distributions give rise to the network effect of GABA, GABA agonists and gap junction blockers. Finally, based on our observations, we suggest that the earliest form of synchronous neuronal activity depends on gap junctions and a decrease in GABA induced depolarization but not on GABAA mediated signaling. PMID:29559893

  4. Human embryonic stem cell-derived neurons adopt and regulate the activity of an established neural network

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    Weick, Jason P.; Liu, Yan; Zhang, Su-Chun

    2011-01-01

    Whether hESC-derived neurons can fully integrate with and functionally regulate an existing neural network remains unknown. Here, we demonstrate that hESC-derived neurons receive unitary postsynaptic currents both in vitro and in vivo and adopt the rhythmic firing behavior of mouse cortical networks via synaptic integration. Optical stimulation of hESC-derived neurons expressing Channelrhodopsin-2 elicited both inhibitory and excitatory postsynaptic currents and triggered network bursting in mouse neurons. Furthermore, light stimulation of hESC-derived neurons transplanted to the hippocampus of adult mice triggered postsynaptic currents in host pyramidal neurons in acute slice preparations. Thus, hESC-derived neurons can participate in and modulate neural network activity through functional synaptic integration, suggesting they are capable of contributing to neural network information processing both in vitro and in vivo. PMID:22106298

  5. Tumor tropism of intravenously injected human-induced pluripotent stem cell-derived neural stem cells and their gene therapy application in a metastatic breast cancer model.

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    Yang, Jing; Lam, Dang Hoang; Goh, Sally Sallee; Lee, Esther Xingwei; Zhao, Ying; Tay, Felix Chang; Chen, Can; Du, Shouhui; Balasundaram, Ghayathri; Shahbazi, Mohammad; Tham, Chee Kian; Ng, Wai Hoe; Toh, Han Chong; Wang, Shu

    2012-05-01

    Human pluripotent stem cells can serve as an accessible and reliable source for the generation of functional human cells for medical therapies. In this study, we used a conventional lentiviral transduction method to derive human-induced pluripotent stem (iPS) cells from primary human fibroblasts and then generated neural stem cells (NSCs) from the iPS cells. Using a dual-color whole-body imaging technology, we demonstrated that after tail vein injection, these human NSCs displayed a robust migratory capacity outside the central nervous system in both immunodeficient and immunocompetent mice and homed in on established orthotopic 4T1 mouse mammary tumors. To investigate whether the iPS cell-derived NSCs can be used as a cellular delivery vehicle for cancer gene therapy, the cells were transduced with a baculoviral vector containing the herpes simplex virus thymidine kinase suicide gene and injected through tail vein into 4T1 tumor-bearing mice. The transduced NSCs were effective in inhibiting the growth of the orthotopic 4T1 breast tumor and the metastatic spread of the cancer cells in the presence of ganciclovir, leading to prolonged survival of the tumor-bearing mice. The use of iPS cell-derived NSCs for cancer gene therapy bypasses the sensitive ethical issue surrounding the use of cells derived from human fetal tissues or human embryonic stem cells. This approach may also help to overcome problems associated with allogeneic transplantation of other types of human NSCs. Copyright © 2012 AlphaMed Press.

  6. The Postischemic Environment Differentially Impacts Teratoma or Tumor Formation After Transplantation of Human Embryonic Stem Cell-Derived Neural Progenitors

    Czech Academy of Sciences Publication Activity Database

    Seminatore, CH.; Polentes, J.; Ellman, D.; Kozubenko, Nataliya; Itier, V.; Tine, S.; Tritschler, L.; Brenot, M.; Guidou, E.; Blondeau, J.; Lhuillier, M.; Bugi, A.; Aubry, L.; Jendelová, Pavla; Syková, Eva; Perrier, A. L.; Finsen, B.; Onteniente, B.

    2010-01-01

    Roč. 41, č. 1 (2010), s. 153-159 ISSN 0039-2499 Institutional research plan: CEZ:AV0Z50390703 Keywords : brain transplantation * human embryonic stem cells * neural differentiation Subject RIV: FH - Neurology Impact factor: 5.756, year: 2010

  7. Quantitative Live Imaging of Human Embryonic Stem Cell Derived Neural Rosettes Reveals Structure-Function Dynamics Coupled to Cortical Development.

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    Ziv, Omer; Zaritsky, Assaf; Yaffe, Yakey; Mutukula, Naresh; Edri, Reuven; Elkabetz, Yechiel

    2015-10-01

    Neural stem cells (NSCs) are progenitor cells for brain development, where cellular spatial composition (cytoarchitecture) and dynamics are hypothesized to be linked to critical NSC capabilities. However, understanding cytoarchitectural dynamics of this process has been limited by the difficulty to quantitatively image brain development in vivo. Here, we study NSC dynamics within Neural Rosettes--highly organized multicellular structures derived from human pluripotent stem cells. Neural rosettes contain NSCs with strong epithelial polarity and are expected to perform apical-basal interkinetic nuclear migration (INM)--a hallmark of cortical radial glial cell development. We developed a quantitative live imaging framework to characterize INM dynamics within rosettes. We first show that the tendency of cells to follow the INM orientation--a phenomenon we referred to as radial organization, is associated with rosette size, presumably via mechanical constraints of the confining structure. Second, early forming rosettes, which are abundant with founder NSCs and correspond to the early proliferative developing cortex, show fast motions and enhanced radial organization. In contrast, later derived rosettes, which are characterized by reduced NSC capacity and elevated numbers of differentiated neurons, and thus correspond to neurogenesis mode in the developing cortex, exhibit slower motions and decreased radial organization. Third, later derived rosettes are characterized by temporal instability in INM measures, in agreement with progressive loss in rosette integrity at later developmental stages. Finally, molecular perturbations of INM by inhibition of actin or non-muscle myosin-II (NMII) reduced INM measures. Our framework enables quantification of cytoarchitecture NSC dynamics and may have implications in functional molecular studies, drug screening, and iPS cell-based platforms for disease modeling.

  8. Quantitative Live Imaging of Human Embryonic Stem Cell Derived Neural Rosettes Reveals Structure-Function Dynamics Coupled to Cortical Development.

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    Omer Ziv

    2015-10-01

    Full Text Available Neural stem cells (NSCs are progenitor cells for brain development, where cellular spatial composition (cytoarchitecture and dynamics are hypothesized to be linked to critical NSC capabilities. However, understanding cytoarchitectural dynamics of this process has been limited by the difficulty to quantitatively image brain development in vivo. Here, we study NSC dynamics within Neural Rosettes--highly organized multicellular structures derived from human pluripotent stem cells. Neural rosettes contain NSCs with strong epithelial polarity and are expected to perform apical-basal interkinetic nuclear migration (INM--a hallmark of cortical radial glial cell development. We developed a quantitative live imaging framework to characterize INM dynamics within rosettes. We first show that the tendency of cells to follow the INM orientation--a phenomenon we referred to as radial organization, is associated with rosette size, presumably via mechanical constraints of the confining structure. Second, early forming rosettes, which are abundant with founder NSCs and correspond to the early proliferative developing cortex, show fast motions and enhanced radial organization. In contrast, later derived rosettes, which are characterized by reduced NSC capacity and elevated numbers of differentiated neurons, and thus correspond to neurogenesis mode in the developing cortex, exhibit slower motions and decreased radial organization. Third, later derived rosettes are characterized by temporal instability in INM measures, in agreement with progressive loss in rosette integrity at later developmental stages. Finally, molecular perturbations of INM by inhibition of actin or non-muscle myosin-II (NMII reduced INM measures. Our framework enables quantification of cytoarchitecture NSC dynamics and may have implications in functional molecular studies, drug screening, and iPS cell-based platforms for disease modeling.

  9. Beneficial effect of human induced pluripotent stem cell-derived neural precursors in spinal cord injury repair

    Czech Academy of Sciences Publication Activity Database

    Romanyuk, Nataliya; Amemori, Takashi; Turnovcová, Karolína; Procházka, Pavel; Onteniente, B.; Syková, Eva; Jendelová, Pavla

    2015-01-01

    Roč. 24, č. 9 (2015), s. 1781-1797 ISSN 0963-6897 R&D Projects: GA MŠk LH12024; GA ČR(CZ) GA13-00939S; GA ČR(CZ) GBP304/12/G069; GA MŠk(CZ) ED1.1.00/02.0109; GA MŠk(CZ) LO1309 Institutional support: RVO:68378041 Keywords : human induced pluripotent stem cells * neural precursors * spinal cord injury Subject RIV: FH - Neurology Impact factor: 3.427, year: 2015

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

    Czech Academy of Sciences Publication Activity Database

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

    2016-01-01

    Roč. 11, č. 2016 (2016), s. 6267-6281 E-ISSN 1178-2013 R&D Projects: GA MŠk(CZ) LQ1604; GA MŠk(CZ) ED1.1.00/02.0109; GA MŠk(CZ) 7F14057 Institutional support: RVO:68378041 ; RVO:61389013 ; RVO:68378271 Keywords : neural precursors * magnetic resonance imaging * cell differentiation Subject RIV: FH - Neurology; EB - Genetics ; Molecular Biology (FGU-C); CD - Macromolecular Chemistry (UMCH-V) Impact factor: 4.300, year: 2016

  11. Transcriptional profiling of MEF2-regulated genes in human neural progenitor cells derived from embryonic stem cells

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    Shing Fai Chan

    2015-03-01

    Full Text Available The myocyte enhancer factor 2 (MEF2 family of transcription factors is highly expressed in the brain and constitutes a key determinant of neuronal survival, differentiation, and synaptic plasticity. However, genome-wide transcriptional profiling of MEF2-regulated genes has not yet been fully elucidated, particularly at the neural stem cell stage. Here we report the results of microarray analysis comparing mRNAs isolated from human neural progenitor/stem cells (hNPCs derived from embryonic stem cells expressing a control vector versus progenitors expressing a constitutively-active form of MEF2 (MEF2CA, which increases MEF2 activity. Microarray experiments were performed using the Illumina Human HT-12 V4.0 expression beadchip (GEO#: GSE57184. By comparing vector-control cells to MEF2CA cells, microarray analysis identified 1880 unique genes that were differentially expressed. Among these genes, 1121 genes were up-regulated and 759 genes were down-regulated. Our results provide a valuable resource for identifying transcriptional targets of MEF2 in hNPCs.

  12. Blastema cells derived from New Zealand white rabbit's pinna carry stemness properties as shown by differentiation into insulin producing, neural, and osteogenic lineages representing three embryonic germ layers.

    Science.gov (United States)

    Saeinasab, Morvarid; Matin, Maryam M; Rassouli, Fatemeh B; Bahrami, Ahmad Reza

    2016-05-01

    Stem cells (SCs) are known as undifferentiated cells with self-renewal and differentiation capacities. Regeneration is a phenomenon that occurs in a limited number of animals after injury, during which blastema tissue is formed. It has been hypothesized that upon injury, the dedifferentiation of surrounding tissues leads into the appearance of cells with SC characteristics. In present study, stem-like cells (SLCs) were obtained from regenerating tissue of New Zealand white rabbit's pinna and their stemness properties were examined by their capacity to differentiate toward insulin producing cells (IPCs), as well as neural and osteogenic lineages. Differentiation was induced by culture of SLCs in defined medium, and cell fates were monitored by specific staining, RT-PCR and flow cytometry assays. Our results revealed that dithizone positive cells, which represent IPCs, and islet-like structures appeared 1 week after induction of SLCs, and this observation was confirmed by the elevated expression of Ins, Pax6 and Glut4 at mRNA level. Furthermore, SLCs were able to express neural markers as early as 1 week after retinoic acid treatment. Finally, SLCs were able to differentiate into osteogenic lineage, as confirmed by Alizarin Red S staining and RT-PCR studies. In conclusion, SLCs, which could successfully differentiate into cells derived from all three germ layers, can be considered as a valuable model to study developmental biology and regenerative medicine.

  13. Safe and efficient method for cryopreservation of human induced pluripotent stem cell-derived neural stem and progenitor cells by a programmed freezer with a magnetic field.

    Science.gov (United States)

    Nishiyama, Yuichiro; Iwanami, Akio; Kohyama, Jun; Itakura, Go; Kawabata, Soya; Sugai, Keiko; Nishimura, Soraya; Kashiwagi, Rei; Yasutake, Kaori; Isoda, Miho; Matsumoto, Morio; Nakamura, Masaya; Okano, Hideyuki

    2016-06-01

    Stem cells represent a potential cellular resource in the development of regenerative medicine approaches to the treatment of pathologies in which specific cells are degenerated or damaged by genetic abnormality, disease, or injury. Securing sufficient supplies of cells suited to the demands of cell transplantation, however, remains challenging, and the establishment of safe and efficient cell banking procedures is an important goal. Cryopreservation allows the storage of stem cells for prolonged time periods while maintaining them in adequate condition for use in clinical settings. Conventional cryopreservation systems include slow-freezing and vitrification both have advantages and disadvantages in terms of cell viability and/or scalability. In the present study, we developed an advanced slow-freezing technique using a programmed freezer with a magnetic field called Cells Alive System (CAS) and examined its effectiveness on human induced pluripotent stem cell-derived neural stem/progenitor cells (hiPSC-NS/PCs). This system significantly increased cell viability after thawing and had less impact on cellular proliferation and differentiation. We further found that frozen-thawed hiPSC-NS/PCs were comparable with non-frozen ones at the transcriptome level. Given these findings, we suggest that the CAS is useful for hiPSC-NS/PCs banking for clinical uses involving neural disorders and may open new avenues for future regenerative medicine. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  14. Pdx1 and Ngn3 overexpression enhances pancreatic differentiation of mouse ES cell-derived endoderm population.

    Science.gov (United States)

    Kubo, Atsushi; Stull, Robert; Takeuchi, Mitsuaki; Bonham, Kristina; Gouon-Evans, Valerie; Sho, Masayuki; Iwano, Masayuki; Saito, Yoshihiko; Keller, Gordon; Snodgrass, Ralph

    2011-01-01

    In order to define the molecular mechanisms regulating the specification and differentiation of pancreatic β-islet cells, we investigated the effect of upregulating Pdx1 and Ngn3 during the differentiation of the β-islet-like cells from murine embryonic stem (ES) cell-derived activin induced-endoderm. Induced overexpression of Pdx1 resulted in a significant upregulation of insulin (Ins1 and Ins2), and other pancreas-related genes. To enhance the developmental progression from the pancreatic bud to the formation of the endocrine lineages, we induced the overexpression express of Ngn3 together with Pdx1. This combination dramatically increased the level and timing of maximal Ins1 mRNA expression to approximately 100% of that found in the βTC6 insulinoma cell line. Insulin protein and C-peptide expression was confirmed by immunohistochemistry staining. These inductive effects were restricted to c-kit(+) endoderm enriched EB-derived populations suggesting that Pdx1/Ngn3 functions after the specification of pancreatic endoderm. Although insulin secretion was stimulated by various insulin secretagogues, these cells had only limited glucose response. Microarray analysis was used to evaluate the expression of a broad spectrum of pancreatic endocrine cell-related genes as well as genes associated with glucose responses. Taken together, these findings demonstrate the utility of manipulating Pdx1 and Ngn3 expression in a stage-specific manner as an important new strategy for the efficient generation of functionally immature insulin-producing β-islet cells from ES cells.

  15. Pdx1 and Ngn3 overexpression enhances pancreatic differentiation of mouse ES cell-derived endoderm population.

    Directory of Open Access Journals (Sweden)

    Atsushi Kubo

    Full Text Available In order to define the molecular mechanisms regulating the specification and differentiation of pancreatic β-islet cells, we investigated the effect of upregulating Pdx1 and Ngn3 during the differentiation of the β-islet-like cells from murine embryonic stem (ES cell-derived activin induced-endoderm. Induced overexpression of Pdx1 resulted in a significant upregulation of insulin (Ins1 and Ins2, and other pancreas-related genes. To enhance the developmental progression from the pancreatic bud to the formation of the endocrine lineages, we induced the overexpression express of Ngn3 together with Pdx1. This combination dramatically increased the level and timing of maximal Ins1 mRNA expression to approximately 100% of that found in the βTC6 insulinoma cell line. Insulin protein and C-peptide expression was confirmed by immunohistochemistry staining. These inductive effects were restricted to c-kit(+ endoderm enriched EB-derived populations suggesting that Pdx1/Ngn3 functions after the specification of pancreatic endoderm. Although insulin secretion was stimulated by various insulin secretagogues, these cells had only limited glucose response. Microarray analysis was used to evaluate the expression of a broad spectrum of pancreatic endocrine cell-related genes as well as genes associated with glucose responses. Taken together, these findings demonstrate the utility of manipulating Pdx1 and Ngn3 expression in a stage-specific manner as an important new strategy for the efficient generation of functionally immature insulin-producing β-islet cells from ES cells.

  16. Gene expression profiling analysis of the effects of low-intensity pulsed ultrasound on induced pluripotent stem cell-derived neural crest stem cells.

    Science.gov (United States)

    Xia, Bin; Zou, Yang; Xu, Zhiling; Lv, Yonggang

    2017-11-01

    Low-intensity pulsed ultrasound (LIPUS) is a noninvasive technique that has been shown to affect cell proliferation, migration, and differentiation and promote the regeneration of damaged peripheral nerve. Our previous studies had proved that LIPUS can significantly promote the neural differentiation of induced pluripotent stem cell-derived neural crest stem cells (iPSCs-NCSCs) and enhance the repair of rat-transected sciatic nerve. To further explore the underlying mechanisms of LIPUS treatment of iPSCs-NCSCs, this study reported the gene expression profiling analysis of iPSCs-NCSCs before and after LIPUS treatment using the RNA-sequencing (RNA-Seq) method. It was found that expression of 76 genes of iPSCs-NCSCs cultured in a serum-free neural induction medium and expression of 21 genes of iPSCs-NCSCs cultured in a neuronal differentiation medium were significantly changed by LIPUS treatment. The differentially expressed genes are related to angiogenesis, nervous system activity and functions, cell activities, and so on. The RNA-seq results were further verified by a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). High correlation was observed between the results obtained from qRT-PCR and RNA-Seq. This study presented new information on the global gene expression patterns of iPSCs-NCSCs after LIPUS treatment and may expand the understanding of the complex molecular mechanism of LIPUS treatment of iPSCs-NCSCs. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

  17. Neuroprotective effect of transplanted human embryonic stem cell-derived neural precursors in an animal model of multiple sclerosis.

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    Michal Aharonowiz

    Full Text Available BACKGROUND: Multiple sclerosis (MS is an immune mediated demyelinating disease of the central nervous system (CNS. A potential new therapeutic approach for MS is cell transplantation which may promote remyelination and suppress the inflammatory process. METHODS: We transplanted human embryonic stem cells (hESC-derived early multipotent neural precursors (NPs into the brain ventricles of mice induced with experimental autoimmune encephalomyelitis (EAE, the animal model of MS. We studied the effect of the transplanted NPs on the functional and pathological manifestations of the disease. RESULTS: Transplanted hESC-derived NPs significantly reduced the clinical signs of EAE. Histological examination showed migration of the transplanted NPs to the host white matter, however, differentiation to mature oligodendrocytes and remyelination were negligible. Time course analysis of the evolution and progression of CNS inflammation and tissue injury showed an attenuation of the inflammatory process in transplanted animals, which was correlated with the reduction of both axonal damage and demyelination. Co-culture experiments showed that hESC-derived NPs inhibited the activation and proliferation of lymph node-derived T cells in response to nonspecific polyclonal stimuli. CONCLUSIONS: The therapeutic effect of transplantation was not related to graft or host remyelination but was mediated by an immunosuppressive neuroprotective mechanism. The attenuation of EAE by hESC-derived NPs, demonstrated here, may serve as the first step towards further developments of hESC for cell therapy in MS.

  18. Differentiated cells derived from fetal neural stem cells improve motor deficits in a rat model of Parkinson’s disease

    Institute of Scientific and Technical Information of China (English)

    Wei Wang; Hao Song; Aifang Shen; Chao Chen; Yanming Liu; Yabing Dong; Fabin Han

    2015-01-01

    Objective: Parkinson’s disease(PD), which is one of the most common neuro‐degenerative disorders, is characterized by the loss of dopamine(DA) neurons in the substantia nigra in the midbrain. Experimental and clinical studies have shown that fetal neural stem cells(NSCs) have therapeutic effects in neurological disorders. The aim of this study was to examine whether cells that were differentiated from NSCs had therapeutic effects in a rat model of PD. Methods: NSCs were isolated from 14‐week‐old embryos and induced to differentiate into neurons, DA neurons, and glial cells, and these cells were characterized by their expression of the following markers: βⅢ‐tubulin and microtubule‐associated protein 2(neurons), tyrosine hydroxylase(DA neurons), and glial fibrillary acidic protein(glial cells). After a 6‐hydroxydopamine(6‐OHDA)‐lesioned rat model of PD was generated, the differentiated cells were transplanted into the striata of the 6‐OHDA‐lesioned PD rats. Results: The motor behaviors of the PD rats were assessed by the number of apomorphine‐induced rotation turns. The results showed that the NSCs differentiated in vitro into neurons and DA neurons with high efficiencies. After transplantation into the striata of the PD rats, the differentiated cells significantly improved the motor deficits of the transplanted PD rats compared to those of the control nontransplanted PD rats by decreasing the apomorphine‐induced turn cycles as early as 4 weeks after transplantation. Immunofluorescence analyses showed that the differentiated DA neurons survived more than 16 weeks. Conclusions: Our results showed that cells that were differentiated from NSCs had therapeutic effects in a rat PD model, which suggests that differentiated cells may be an effective treatment for patients with PD.

  19. Engrafted human induced pluripotent stem cell-derived anterior specified neural progenitors protect the rat crushed optic nerve.

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    Leila Satarian

    Full Text Available BACKGROUND: Degeneration of retinal ganglion cells (RGCs is a common occurrence in several eye diseases. This study examined the functional improvement and protection of host RGCs in addition to the survival, integration and neuronal differentiation capabilities of anterior specified neural progenitors (NPs following intravitreal transplantation. METHODOLOGY/PRINCIPAL FINDINGS: NPs were produced under defined conditions from human induced pluripotent stem cells (hiPSCs and transplanted into rats whose optic nerves have been crushed (ONC. hiPSCs were induced to differentiate into anterior specified NPs by the use of Noggin and retinoic acid. The hiPSC-NPs were labeled by green fluorescent protein or a fluorescent tracer 1,1' -dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI and injected two days after induction of ONC in hooded rats. Functional analysis according to visual evoked potential recordings showed significant amplitude recovery in animals transplanted with hiPSC-NPs. Retrograde labeling by an intra-collicular DiI injection showed significantly higher numbers of RGCs and spared axons in ONC rats treated with hiPSC-NPs or their conditioned medium (CM. The analysis of CM of hiPSC-NPs showed the secretion of ciliary neurotrophic factor, basic fibroblast growth factor, and insulin-like growth factor. Optic nerve of cell transplanted groups also had increased GAP43 immunoreactivity and myelin staining by FluoroMyelin™ which imply for protection of axons and myelin. At 60 days post-transplantation hiPSC-NPs were integrated into the ganglion cell layer of the retina and expressed neuronal markers. CONCLUSIONS/SIGNIFICANCE: The transplantation of anterior specified NPs may improve optic nerve injury through neuroprotection and differentiation into neuronal lineages. These NPs possibly provide a promising new therapeutic approach for traumatic optic nerve injuries and loss of RGCs caused by other diseases.

  20. Rapid generation of sub-type, region-specific neurons and neural networks from human pluripotent stem cell-derived neurospheres

    Directory of Open Access Journals (Sweden)

    Aynun N. Begum

    2015-11-01

    Full Text Available Stem cell-based neuronal differentiation has provided a unique opportunity for disease modeling and regenerative medicine. Neurospheres are the most commonly used neuroprogenitors for neuronal differentiation, but they often clump in culture, which has always represented a challenge for neurodifferentiation. In this study, we report a novel method and defined culture conditions for generating sub-type or region-specific neurons from human embryonic and induced pluripotent stem cells derived neurosphere without any genetic manipulation. Round and bright-edged neurospheres were generated in a supplemented knockout serum replacement medium (SKSRM with 10% CO2, which doubled the expression of the NESTIN, PAX6 and FOXG1 genes compared with those cultured with 5% CO2. Furthermore, an additional step (AdSTEP was introduced to fragment the neurospheres and facilitate the formation of a neuroepithelial-type monolayer that we termed the “neurosphederm”. The large neural tube-type rosette (NTTR structure formed from the neurosphederm, and the NTTR expressed higher levels of the PAX6, SOX2 and NESTIN genes compared with the neuroectoderm-derived neuroprogenitors. Different layers of cortical, pyramidal, GABAergic, glutamatergic, cholinergic neurons appeared within 27 days using the neurosphederm, which is a shorter period than in traditional neurodifferentiation-protocols (42–60 days. With additional supplements and timeline dopaminergic and Purkinje neurons were also generated in culture too. Furthermore, our in vivo results indicated that the fragmented neurospheres facilitated significantly better neurogenesis in severe combined immunodeficiency (SCID mouse brains compared with the non-fragmented neurospheres. Therefore, this neurosphere-based neurodifferentiation protocol is a valuable tool for studies of neurodifferentiation, neuronal transplantation and high throughput screening assays.

  1. Awakened by cellular stress: isolation and characterization of a novel population of pluripotent stem cells derived from human adipose tissue.

    Directory of Open Access Journals (Sweden)

    Saleh Heneidi

    Full Text Available Advances in stem cell therapy face major clinical limitations, particularly challenged by low rates of post-transplant cell survival. Hostile host factors of the engraftment microenvironment such as hypoxia, nutrition deprivation, pro-inflammatory cytokines, and reactive oxygen species can each contribute to unwanted differentiation or apoptosis. In this report, we describe the isolation and characterization of a new population of adipose tissue (AT derived pluripotent stem cells, termed Multilineage Differentiating Stress-Enduring (Muse Cells, which are isolated using severe cellular stress conditions, including long-term exposure to the proteolytic enzyme collagenase, serum deprivation, low temperatures and hypoxia. Under these conditions, a highly purified population of Muse-AT cells is isolated without the utilization of cell sorting methods. Muse-AT cells grow in suspension as cell spheres reminiscent of embryonic stem cell clusters. Muse-AT cells are positive for the pluripotency markers SSEA3, TR-1-60, Oct3/4, Nanog and Sox2, and can spontaneously differentiate into mesenchymal, endodermal and ectodermal cell lineages with an efficiency of 23%, 20% and 22%, respectively. When using specific differentiation media, differentiation efficiency is greatly enhanced in Muse-AT cells (82% for mesenchymal, 75% for endodermal and 78% for ectodermal. When compared to adipose stem cells (ASCs, microarray data indicate a substantial up-regulation of Sox2, Oct3/4, and Rex1. Muse-ATs also exhibit gene expression patterns associated with the down-regulation of genes involved in cell death and survival, embryonic development, DNA replication and repair, cell cycle and potential factors related to oncogenecity. Gene expression analysis indicates that Muse-ATs and ASCs are mesenchymal in origin; however, Muse-ATs also express numerous lymphocytic and hematopoietic genes, such as CCR1 and CXCL2, encoding chemokine receptors and ligands involved in stem cell

  2. The Use of Human-Induced Pluripotent Stem Cell-Derived Neural Precursors in the Treatment of Brain and Spinal Cord Injury

    Czech Academy of Sciences Publication Activity Database

    Jendelová, Pavla; Kozubenko, Nataliya; Amemori, Takashi; Turnovcová, Karolína; Seminatore, CH.; Jirák, D.; Onteniente, B.; Syková, Eva

    2011-01-01

    Roč. 20, č. 4 (2011), s. 564-564 ISSN 0963-6897. [International Neural Transplantatioin and Repair Meeting/18th Annual Meeting of the American-Society-for- Neural - Therapy - and -Repair /11./. 04.05.2011-08.05.2011, Clearwater] Institutional research plan: CEZ:AV0Z50390703 Keywords : spinal cord * stem cell Subject RIV: FH - Neurology

  3. Integration of donor mesenchymal stem cell-derived neuron-like cells into host neural network after rat spinal cord transection.

    Science.gov (United States)

    Zeng, Xiang; Qiu, Xue-Cheng; Ma, Yuan-Huan; Duan, Jing-Jing; Chen, Yuan-Feng; Gu, Huai-Yu; Wang, Jun-Mei; Ling, Eng-Ang; Wu, Jin-Lang; Wu, Wutian; Zeng, Yuan-Shan

    2015-06-01

    Functional deficits following spinal cord injury (SCI) primarily attribute to loss of neural connectivity. We therefore tested if novel tissue engineering approaches could enable neural network repair that facilitates functional recovery after spinal cord transection (SCT). Rat bone marrow-derived mesenchymal stem cells (MSCs), genetically engineered to overexpress TrkC, receptor of neurotrophin-3 (NT-3), were pre-differentiated into cells carrying neuronal features via co-culture with NT-3 overproducing Schwann cells in 3-dimensional gelatin sponge (GS) scaffold for 14 days in vitro. Intra-GS formation of MSC assemblies emulating neural network (MSC-GS) were verified morphologically via electron microscopy (EM) and functionally by whole-cell patch clamp recording of spontaneous post-synaptic currents. The differentiated MSCs still partially maintained prototypic property with the expression of some mesodermal cytokines. MSC-GS or GS was then grafted acutely into a 2 mm-wide transection gap in the T9-T10 spinal cord segments of adult rats. Eight weeks later, hindlimb function of the MSC-GS-treated SCT rats was significantly improved relative to controls receiving the GS or lesion only as indicated by BBB score. The MSC-GS transplantation also significantly recovered cortical motor evoked potential (CMEP). Histologically, MSC-derived neuron-like cells maintained their synapse-like structures in vivo; they additionally formed similar connections with host neurites (i.e., mostly serotonergic fibers plus a few corticospinal axons; validated by double-labeled immuno-EM). Moreover, motor cortex electrical stimulation triggered c-fos expression in the grafted and lumbar spinal cord cells of the treated rats only. Our data suggest that MSC-derived neuron-like cells resulting from NT-3-TrkC-induced differentiation can partially integrate into transected spinal cord and this strategy should be further investigated for reconstructing disrupted neural circuits. Copyright

  4. Integrative analysis of genes and miRNA alterations in human embryonic stem cells-derived neural cells after exposure to silver nanoparticles

    International Nuclear Information System (INIS)

    Oh, Jung-Hwa; Son, Mi-Young; Choi, Mi-Sun; Kim, Soojin; Choi, A-young; Lee, Hyang-Ae; Kim, Ki-Suk; Kim, Janghwan; Song, Chang Woo; Yoon, Seokjoo

    2016-01-01

    Given the rapid growth of engineered and customer products made of silver nanoparticles (Ag NPs), understanding their biological and toxicological effects on humans is critically important. The molecular developmental neurotoxic effects associated with exposure to Ag NPs were analyzed at the physiological and molecular levels, using an alternative cell model: human embryonic stem cell (hESC)-derived neural stem/progenitor cells (NPCs). In this study, the cytotoxic effects of Ag NPs (10–200 μg/ml) were examined in these hESC-derived NPCs, which have a capacity for neurogenesis in vitro, at 6 and 24 h. The results showed that Ag NPs evoked significant toxicity in hESC-derived NPCs at 24 h in a dose-dependent manner. In addition, Ag NPs induced cell cycle arrest and apoptosis following a significant increase in oxidative stress in these cells. To further clarify the molecular mechanisms of the toxicological effects of Ag NPs at the transcriptional and post-transcriptional levels, the global expression profiles of genes and miRNAs were analyzed in hESC-derived NPCs after Ag NP exposure. The results showed that Ag NPs induced oxidative stress and dysfunctional neurogenesis at the molecular level in hESC-derived NPCs. Based on this hESC-derived neural cell model, these findings have increased our understanding of the molecular events underlying developmental neurotoxicity induced by Ag NPs in humans. - Highlights: • This system served as a suitable model for developmental neurotoxicity testing. • Ag NPs induce the apoptosis in human neural cells by ROS generation. • Genes for development of neurons were dysregulated in response to Ag NPs. • Molecular events during early developmental neurotoxicity were proposed.

  5. Effects of the Post-Spinal Cord Injury Microenvironment on the Differentiation Capacity of Human Neural Stem Cells Derived from Induced Pluripotent Stem Cells.

    Science.gov (United States)

    López-Serrano, Clara; Torres-Espín, Abel; Hernández, Joaquim; Alvarez-Palomo, Ana B; Requena, Jordi; Gasull, Xavier; Edel, Michael J; Navarro, Xavier

    2016-10-01

    Spinal cord injury (SCI) causes loss of neural functions below the level of the lesion due to interruption of spinal pathways and secondary neurodegenerative processes. The transplant of neural stem cells (NSCs) is a promising approach for the repair of SCI. Reprogramming of adult somatic cells into induced pluripotent stem cells (iPSCs) is expected to provide an autologous source of iPSC-derived NSCs, avoiding the immune response as well as ethical issues. However, there is still limited information on the behavior and differentiation pattern of transplanted iPSC-derived NSCs within the damaged spinal cord. We transplanted iPSC-derived NSCs, obtained from adult human somatic cells, into rats at 0 or 7 days after SCI, and evaluated motor-evoked potentials and locomotion of the animals. We histologically analyzed engraftment, proliferation, and differentiation of the iPSC-derived NSCs and the spared tissue in the spinal cords at 7, 21, and 63 days posttransplant. Both transplanted groups showed a late decline in functional recovery compared to vehicle-injected groups. Histological analysis showed proliferation of transplanted cells within the tissue and that cells formed a mass. At the final time point, most grafted cells differentiated to neural and astroglial lineages, but not into oligodendrocytes, while some grafted cells remained undifferentiated and proliferative. The proinflammatory tissue microenviroment of the injured spinal cord induced proliferation of the grafted cells and, therefore, there are possible risks associated with iPSC-derived NSC transplantation. New approaches are needed to promote and guide cell differentiation, as well as reduce their tumorigenicity once the cells are transplanted at the lesion site.

  6. Integrative analysis of genes and miRNA alterations in human embryonic stem cells-derived neural cells after exposure to silver nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Oh, Jung-Hwa [Korea Institute of Toxicology (KIT), Daejeon 34114 (Korea, Republic of); Department of human and environmental toxicology, University of Science & Technology, Daejeon 34113 (Korea, Republic of); Son, Mi-Young [Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahangno, Yuseong-gu, Daejeon 34141 (Korea, Republic of); Department of functional genomics, University of Science & Technology, 217 Gajungro, Yuseong-gu, Daejeon 34113 (Korea, Republic of); Choi, Mi-Sun; Kim, Soojin; Choi, A-young [Korea Institute of Toxicology (KIT), Daejeon 34114 (Korea, Republic of); Lee, Hyang-Ae; Kim, Ki-Suk [Korea Institute of Toxicology (KIT), Daejeon 34114 (Korea, Republic of); Department of human and environmental toxicology, University of Science & Technology, Daejeon 34113 (Korea, Republic of); Kim, Janghwan [Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahangno, Yuseong-gu, Daejeon 34141 (Korea, Republic of); Department of functional genomics, University of Science & Technology, 217 Gajungro, Yuseong-gu, Daejeon 34113 (Korea, Republic of); Song, Chang Woo, E-mail: cwsong@kitox.re.kr [Korea Institute of Toxicology (KIT), Daejeon 34114 (Korea, Republic of); Department of human and environmental toxicology, University of Science & Technology, Daejeon 34113 (Korea, Republic of); Yoon, Seokjoo, E-mail: sjyoon@kitox.re.kr [Korea Institute of Toxicology (KIT), Daejeon 34114 (Korea, Republic of); Department of human and environmental toxicology, University of Science & Technology, Daejeon 34113 (Korea, Republic of)

    2016-05-15

    Given the rapid growth of engineered and customer products made of silver nanoparticles (Ag NPs), understanding their biological and toxicological effects on humans is critically important. The molecular developmental neurotoxic effects associated with exposure to Ag NPs were analyzed at the physiological and molecular levels, using an alternative cell model: human embryonic stem cell (hESC)-derived neural stem/progenitor cells (NPCs). In this study, the cytotoxic effects of Ag NPs (10–200 μg/ml) were examined in these hESC-derived NPCs, which have a capacity for neurogenesis in vitro, at 6 and 24 h. The results showed that Ag NPs evoked significant toxicity in hESC-derived NPCs at 24 h in a dose-dependent manner. In addition, Ag NPs induced cell cycle arrest and apoptosis following a significant increase in oxidative stress in these cells. To further clarify the molecular mechanisms of the toxicological effects of Ag NPs at the transcriptional and post-transcriptional levels, the global expression profiles of genes and miRNAs were analyzed in hESC-derived NPCs after Ag NP exposure. The results showed that Ag NPs induced oxidative stress and dysfunctional neurogenesis at the molecular level in hESC-derived NPCs. Based on this hESC-derived neural cell model, these findings have increased our understanding of the molecular events underlying developmental neurotoxicity induced by Ag NPs in humans. - Highlights: • This system served as a suitable model for developmental neurotoxicity testing. • Ag NPs induce the apoptosis in human neural cells by ROS generation. • Genes for development of neurons were dysregulated in response to Ag NPs. • Molecular events during early developmental neurotoxicity were proposed.

  7. Protease-activated receptor-1 negatively regulates proliferation of neural stem/progenitor cells derived from the hippocampal dentate gyrus of the adult mouse

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    Masayuki Tanaka

    2016-07-01

    Full Text Available Thrombin-activated protease-activated receptor (PAR-1 regulates the proliferation of neural cells following brain injury. To elucidate the involvement of PAR-1 in the neurogenesis that occurs in the adult hippocampus, we examined whether PAR-1 regulated the proliferation of neural stem/progenitor cells (NPCs derived from the murine hippocampal dentate gyrus. NPC cultures expressed PAR-1 protein and mRNA encoding all subtypes of PAR. Direct exposure of the cells to thrombin dramatically attenuated the cell proliferation without causing cell damage. This thrombin-induced attenuation was almost completely abolished by the PAR antagonist RWJ 56110, as well as by dabigatran and 4-(2-aminoethylbenzenesulfonyl fluoride (AEBSF, which are selective and non-selective thrombin inhibitors, respectively. Expectedly, the PAR-1 agonist peptide (AP SFLLR-NH2 also attenuated the cell proliferation. The cell proliferation was not affected by the PAR-1 negative control peptide RLLFT-NH2, which is an inactive peptide for PAR-1. Independently, we determined the effect of in vivo treatment with AEBSF or AP on hippocampal neurogenesis in the adult mouse. The administration of AEBSF, but not that of AP, significantly increased the number of newly-generated cells in the hippocampal subgranular zone. These data suggest that PAR-1 negatively regulated adult neurogenesis in the hippocampus by inhibiting the proliferative activity of the NPCs.

  8. A simple, xeno-free method for oligodendrocyte generation from human neural stem cells derived from umbilical cord: engagement of gelatinases in cell commitment and differentiation.

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    Sypecka, Joanna; Ziemka-Nalecz, Małgorzata; Dragun-Szymczak, Patrycja; Zalewska, Teresa

    2017-05-01

    Oligodendrocyte progenitors (OPCs) are ranked among the most likely candidates for cell-based strategies aimed at treating neurodegenerative diseases accompanied by dys/demyelination of the central nervous system (CNS). In this regard, different sources of stem cells are being tested to elaborate xeno-free protocols for efficient generation of OPCs for clinical applications. In the present study, neural stem cells of human umbilical cord blood (HUCB-NSCs) have been used to derive OPCs and subsequently to differentiate them into mature, GalC-expressing oligodendrocytes. Applied components of the extracellular matrix (ECM) and the analogues of physiological substances known to increase glial commitment of neural stem cells have been shown to significantly increase the yield of the resulting OPC fraction. The efficiency of ECM components in promoting oligodendrocyte commitment and differentiation prompted us to investigate the potential role of gelatinases in those processes. Subsequently, endogenous and ECM metalloproteinases (MMPs) activity has been compared with that detected in primary cultures of rat oligodendrocytes in vitro, as well as in rat brains in vivo. The data indicate that gelatinases are engaged in gliogenesis both in vitro and in vivo, although differently, which presumably results from distinct extracellular conditions. In conclusion, the study presents an efficient xeno-free method of deriving oligodendrocyte from HUCB-NSCs and analyses the engagement of MMP-2/MMP-9 in the processes of cell commitment and maturation. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  9. A Perturbed MicroRNA Expression Pattern Characterizes Embryonic Neural Stem Cells Derived from a Severe Mouse Model of Spinal Muscular Atrophy (SMA

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    Andrea Luchetti

    2015-08-01

    Full Text Available Spinal muscular atrophy (SMA is an inherited neuromuscular disorder and the leading genetic cause of death in infants. Despite the disease-causing gene, survival motor neuron (SMN1, encodes a ubiquitous protein, SMN1 deficiency preferentially affects spinal motor neurons (MNs, leaving the basis of this selective cell damage still unexplained. As neural stem cells (NSCs are multipotent self-renewing cells that can differentiate into neurons, they represent an in vitro model for elucidating the pathogenetic mechanism of neurodegenerative diseases such as SMA. Here we characterize for the first time neural stem cells (NSCs derived from embryonic spinal cords of a severe SMNΔ7 SMA mouse model. SMNΔ7 NSCs behave as their wild type (WT counterparts, when we consider neurosphere formation ability and the expression levels of specific regional and self-renewal markers. However, they show a perturbed cell cycle phase distribution and an increased proliferation rate compared to wild type cells. Moreover, SMNΔ7 NSCs are characterized by the differential expression of a limited number of miRNAs, among which miR-335-5p and miR-100-5p, reduced in SMNΔ7 NSCs compared to WT cells. We suggest that such miRNAs may be related to the proliferation differences characterizing SMNΔ7 NSCs, and may be potentially involved in the molecular mechanisms of SMA.

  10. A Perturbed MicroRNA Expression Pattern Characterizes Embryonic Neural Stem Cells Derived from a Severe Mouse Model of Spinal Muscular Atrophy (SMA).

    Science.gov (United States)

    Luchetti, Andrea; Ciafrè, Silvia Anna; Murdocca, Michela; Malgieri, Arianna; Masotti, Andrea; Sanchez, Massimo; Farace, Maria Giulia; Novelli, Giuseppe; Sangiuolo, Federica

    2015-08-06

    Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder and the leading genetic cause of death in infants. Despite the disease-causing gene, survival motor neuron (SMN1), encodes a ubiquitous protein, SMN1 deficiency preferentially affects spinal motor neurons (MNs), leaving the basis of this selective cell damage still unexplained. As neural stem cells (NSCs) are multipotent self-renewing cells that can differentiate into neurons, they represent an in vitro model for elucidating the pathogenetic mechanism of neurodegenerative diseases such as SMA. Here we characterize for the first time neural stem cells (NSCs) derived from embryonic spinal cords of a severe SMNΔ7 SMA mouse model. SMNΔ7 NSCs behave as their wild type (WT) counterparts, when we consider neurosphere formation ability and the expression levels of specific regional and self-renewal markers. However, they show a perturbed cell cycle phase distribution and an increased proliferation rate compared to wild type cells. Moreover, SMNΔ7 NSCs are characterized by the differential expression of a limited number of miRNAs, among which miR-335-5p and miR-100-5p, reduced in SMNΔ7 NSCs compared to WT cells. We suggest that such miRNAs may be related to the proliferation differences characterizing SMNΔ7 NSCs, and may be potentially involved in the molecular mechanisms of SMA.

  11. Integrative analysis of genes and miRNA alterations in human embryonic stem cells-derived neural cells after exposure to silver nanoparticles.

    Science.gov (United States)

    Oh, Jung-Hwa; Son, Mi-Young; Choi, Mi-Sun; Kim, Soojin; Choi, A-Young; Lee, Hyang-Ae; Kim, Ki-Suk; Kim, Janghwan; Song, Chang Woo; Yoon, Seokjoo

    2016-05-15

    Given the rapid growth of engineered and customer products made of silver nanoparticles (Ag NPs), understanding their biological and toxicological effects on humans is critically important. The molecular developmental neurotoxic effects associated with exposure to Ag NPs were analyzed at the physiological and molecular levels, using an alternative cell model: human embryonic stem cell (hESC)-derived neural stem/progenitor cells (NPCs). In this study, the cytotoxic effects of Ag NPs (10-200μg/ml) were examined in these hESC-derived NPCs, which have a capacity for neurogenesis in vitro, at 6 and 24h. The results showed that Ag NPs evoked significant toxicity in hESC-derived NPCs at 24h in a dose-dependent manner. In addition, Ag NPs induced cell cycle arrest and apoptosis following a significant increase in oxidative stress in these cells. To further clarify the molecular mechanisms of the toxicological effects of Ag NPs at the transcriptional and post-transcriptional levels, the global expression profiles of genes and miRNAs were analyzed in hESC-derived NPCs after Ag NP exposure. The results showed that Ag NPs induced oxidative stress and dysfunctional neurogenesis at the molecular level in hESC-derived NPCs. Based on this hESC-derived neural cell model, these findings have increased our understanding of the molecular events underlying developmental neurotoxicity induced by Ag NPs in humans. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. In vitro evaluation of biocompatibility of uncoated thermally reduced graphene and carbon nanotube-loaded PVDF membranes with adult neural stem cell-derived neurons and glia

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    Çagla Defterali

    2016-12-01

    Full Text Available Graphene, graphene-based nanomaterials (GBNs and carbon nanotubes (CNTs are being investigated as potential substrates for the growth of neural cells. However, in most in vitro studies the cells were seeded on these materials coated with various proteins implying that the observed effects on the cells could not solely be attributed to the GBN and CNT properties. Here we studied the biocompatibility of uncoated thermally reduced graphene (TRG and poly-vinylidene fluoride (PVDF membranes loaded with multi walled CNTs (MWCNTs using neural stem cells (NSCs isolated from the adult mouse olfactory bulb (termed aOBSCs. When aOBSCs were induced to differentiate on coverslips treated with TRG or control materials (polyethyleneimine-PEI and polyornithine plus fibronectin-PLO/F in a serum-free medium, neurons, astrocytes, and oligodendrocytes were generated in all conditions, indicating that TRG permits the multi-lineage differentiation of aOBSCs. However, the total number of cells was reduced on both PEI and TRG. In a serum-containing medium, aOBSC-derived neurons and oligodendrocytes grown on TRG were more numerous than in controls; the neurons developed synaptic boutons and oligodendrocytes were more branched. In contrast, neurons growing on PVDF membranes had reduced neurite branching and on MWCNTs-loaded membranes, oligodendrocytes were lower in numbers than in controls. Overall, these findings indicate that uncoated TRG may be biocompatible with the generation, differentiation, and maturation of aOBSC-derived neurons and glial cells, implying a potential use for TRG to study functional neuronal networks.

  13. Sortilin-Related Receptor Expression in Human Neural Stem Cells Derived from Alzheimer’s Disease Patients Carrying the APOE Epsilon 4 Allele

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    Alen Zollo

    2017-01-01

    Full Text Available Alzheimer’s disease (AD is the most common form of dementia in the elderly; important risk factors are old age and inheritance of the apolipoprotein E4 (APOE4 allele. Changes in amyloid precursor protein (APP binding, trafficking, and sorting may be important AD causative factors. Secretase-mediated APP cleavage produces neurotoxic amyloid-beta (Aβ peptides, which form lethal deposits in the brain. In vivo and in vitro studies have implicated sortilin-related receptor (SORL1 as an important factor in APP trafficking and processing. Recent in vitro evidence has associated the APOE4 allele and alterations in the SORL1 pathway with AD development and progression. Here, we analyzed SORL1 expression in neural stem cells (NSCs from AD patients carrying null, one, or two copies of the APOE4 allele. We show reduced SORL1 expression only in NSCs of a patient carrying two copies of APOE4 allele with increased Aβ/SORL1 localization along the degenerated neurites. Interestingly, SORL1 binding to APP was largely compromised; this could be almost completely reversed by γ-secretase (but not β-secretase inhibitor treatment. These findings may yield new insights into the complex interplay of SORL1 and AD pathology and point to NSCs as a valuable tool to address unsolved AD-related questions in vitro.

  14. Modulation of the major histocompatibility complex by neural stem cell-derived neurotrophic factors used for regenerative therapy in a rat model of stroke

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    Sun Chongran

    2010-08-01

    Full Text Available Abstract Background The relationship between functional improvements in ischemic rats given a neural stem cell (NSC transplant and the modulation of the class I major histocompatibility complex (MHC mediated by NSC-derived neurotrophins was investigated. Methods The levels of gene expression of nerve growth factor (NGF, brain-derived neurotropic factor (BDNF and neurotrophin-3 (NT-3 were assayed from cultures of cortical NSC from Sprague-Dawley rat E16 embryos. The levels of translated NGF in spent culture media from NSC cultures and the cerebral spinal fluid (CSF of rats with and without NGF injection or NSC transplant were also measured. Results We found a significant increase of NGF, BDNF and NT-3 transcripts and NGF proteins in both the NSC cultures and the CSF of the rats. The immunochemical staining for MHC in brain sections and the enzyme-linked immunosorbent assay of CSF were carried out in sham-operated rats and rats with surgically induced focal cerebral ischemia. These groups were further divided into animals that did and did not receive NGF administration or NSC transplant into the cisterna magna. Our results show an up-regulation of class I MHC in the ischemic rats with NGF and NSC administration. The extent of caspase-III immunoreactivity was comparable among three arms in the ischemic rats. Conclusion Readouts of somatosensory evoked potential and the trap channel test illustrated improvements in the neurological function of ischemic rats treated with NGF administration and NSC transplant.

  15. Rotary orbital suspension culture of embryonic stem cell-derived neural stem/progenitor cells: impact of hydrodynamic culture on aggregate yield, morphology and cell phenotype.

    Science.gov (United States)

    Laundos, Tiago L; Silva, Joana; Assunção, Marisa; Quelhas, Pedro; Monteiro, Cátia; Oliveira, Carla; Oliveira, Maria J; Pêgo, Ana P; Amaral, Isabel F

    2017-08-01

    Embryonic stem (ES)-derived neural stem/progenitor cells (ES-NSPCs) constitute a promising cell source for application in cell therapies for the treatment of central nervous system disorders. In this study, a rotary orbital hydrodynamic culture system was applied to single-cell suspensions of ES-NSPCs, to obtain homogeneously-sized ES-NSPC cellular aggregates (neurospheres). Hydrodynamic culture allowed the formation of ES-NSPC neurospheres with a narrower size distribution than statically cultured neurospheres, increasing orbital speeds leading to smaller-sized neurospheres and higher neurosphere yield. Neurospheres formed under hydrodynamic conditions (72 h at 55 rpm) showed higher cell compaction and comparable percentages of viable, dead, apoptotic and proliferative cells. Further characterization of cellular aggregates provided new insights into the effect of hydrodynamic shear on ES-NSPC behaviour. Rotary neurospheres exhibited reduced protein levels of N-cadherin and β-catenin, and higher deposition of laminin (without impacting fibronectin deposition), matrix metalloproteinase-2 (MMP-2) activity and percentage of neuronal cells. In line with the increased MMP-2 activity levels found, hydrodynamically-cultured neurospheres showed higher outward migration on laminin. Moreover, when cultured in a 3D fibrin hydrogel, rotary neurospheres generated an increased percentage of neuronal cells. In conclusion, the application of a constant orbital speed to single-cell suspensions of ES-NSPCs, besides allowing the formation of homogeneously-sized neurospheres, promoted ES-NSPC differentiation and outward migration, possibly by influencing the expression of cell-cell adhesion molecules and the secretion of proteases/extracellular matrix proteins. These findings are important when establishing the culture conditions needed to obtain uniformly-sized ES-NSPC aggregates, either for use in regenerative therapies or in in vitro platforms for biomaterial development or

  16. Creatine Enhances Transdifferentiation of Bone Marrow Stromal Cell-Derived Neural Stem Cell Into GABAergic Neuron-Like Cells Characterized With Differential Gene Expression.

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    Darabi, Shahram; Tiraihi, Taki; Delshad, AliReza; Sadeghizadeh, Majid; Taheri, Taher; Hassoun, Hayder K

    2017-04-01

    Creatine was reported to induce bone marrow stromal cells (BMSC) into GABAergic neuron-like cells (GNLC). In a previous study, creatine was used as a single inducer for BMSC into GNLC with low yield. In this study, BMSC-derived neurospheres (NS) have been used in generating GABAergic phenotype. The BMSC were isolated from adult rats and used in generating neurospheres and used for producing neural stem cells (NSC). A combination of all-trans-retinoic acid (RA), the ciliary neurotrophic factor (CNTF), and creatine was used in order to improve the yield of GNLC. We also used other protocols for the transdifferentiation including RA alone; RA and creatine; RA and CNTF; and RA, CNTF, and creatine. The BMSC, NSC, and GNLC were characterized by specific markers. The activity of the GNLC was evaluated using FM1-43. The isolated BMSC expressed Oct4, fibronectin, and CD44. The NS were immunoreactive to nestin and SOX2, the NSC were immunoreactive to nestin, NF68 and NF160, while the GNLC were immunoreactive to GAD1/2, VGAT, GABA, and synaptophysin. Oct4 and c-MYC, pluripotency genes, were expressed in the BMSC, while SOX2 and c-MYC were expressed in the NSC. The activity of GNLC indicates that the synaptic vesicles were released upon stimulation. The conclusion is that the combination of RA, CNTF, and creatine induced differentiation of neurosphere-derived NSC into GNLC within 1 week. This protocol gives higher yield than the other protocols used in this study. The mechanism of induction was clearly associated with several differential pluripotent genes.

  17. Male-specific differences in proliferation, neurogenesis, and sensitivity to oxidative stress in neural progenitor cells derived from a rat model of ALS.

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    Ruojia Li

    Full Text Available Amyotrophic Lateral Sclerosis (ALS is a fatal neurodegenerative disease characterized by progressive motor dysfunction and the loss of large motor neurons in the spinal cord and brain stem. A clear genetic link to point mutations in the superoxide dismutase 1 (SOD1 gene has been shown in a small group of familial ALS patients. The exact etiology of ALS is still uncertain, but males have consistently been shown to be at a higher risk for the disease than females. Here we present male-specific effects of the mutant SOD1 transgene on proliferation, neurogenesis, and sensitivity to oxidative stress in rat neural progenitor cells (rNPCs. E14 pups were bred using SOD1(G93A transgenic male rats and wild-type female rats. The spinal cord and cortex tissues were collected, genotyped by PCR using primers for the SOD1(G93A transgene or the male-specific Sry gene, and cultured as neurospheres. The number of dividing cells was higher in male rNPCs compared to female rNPCs. However, SOD1(G93A over-expression significantly reduced cell proliferation in male cells but not female cells. Similarly, male rNPCs produced more neurons compared to female rNPCs, but SOD1(G93A over-expression significantly reduced the number of neurons produced in male cells. Finally we asked whether sex and SOD1(G93A transgenes affected sensitivity to oxidative stress. There was no sex-based difference in cell viability after treatment with hydrogen peroxide or 3-morpholinosydnonimine, a free radical-generating agent. However, increased cytotoxicity by SOD1(G93A over-expression occurred, especially in male rNPCs. These results provide essential information on how the mutant SOD1 gene and sexual dimorphism are involved in ALS disease progression.

  18. Stimulus-dependent maximum entropy models of neural population codes.

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    Einat Granot-Atedgi

    Full Text Available Neural populations encode information about their stimulus in a collective fashion, by joint activity patterns of spiking and silence. A full account of this mapping from stimulus to neural activity is given by the conditional probability distribution over neural codewords given the sensory input. For large populations, direct sampling of these distributions is impossible, and so we must rely on constructing appropriate models. We show here that in a population of 100 retinal ganglion cells in the salamander retina responding to temporal white-noise stimuli, dependencies between cells play an important encoding role. We introduce the stimulus-dependent maximum entropy (SDME model-a minimal extension of the canonical linear-nonlinear model of a single neuron, to a pairwise-coupled neural population. We find that the SDME model gives a more accurate account of single cell responses and in particular significantly outperforms uncoupled models in reproducing the distributions of population codewords emitted in response to a stimulus. We show how the SDME model, in conjunction with static maximum entropy models of population vocabulary, can be used to estimate information-theoretic quantities like average surprise and information transmission in a neural population.

  19. Understanding the Implications of Neural Population Activity on Behavior

    Science.gov (United States)

    Briguglio, John

    Learning how neural activity in the brain leads to the behavior we exhibit is one of the fundamental questions in Neuroscience. In this dissertation, several lines of work are presented to that use principles of neural coding to understand behavior. In one line of work, we formulate the efficient coding hypothesis in a non-traditional manner in order to test human perceptual sensitivity to complex visual textures. We find a striking agreement between how variable a particular texture signal is and how sensitive humans are to its presence. This reveals that the efficient coding hypothesis is still a guiding principle for neural organization beyond the sensory periphery, and that the nature of cortical constraints differs from the peripheral counterpart. In another line of work, we relate frequency discrimination acuity to neural responses from auditory cortex in mice. It has been previously observed that optogenetic manipulation of auditory cortex, in addition to changing neural responses, evokes changes in behavioral frequency discrimination. We are able to account for changes in frequency discrimination acuity on an individual basis by examining the Fisher information from the neural population with and without optogenetic manipulation. In the third line of work, we address the question of what a neural population should encode given that its inputs are responses from another group of neurons. Drawing inspiration from techniques in machine learning, we train Deep Belief Networks on fake retinal data and show the emergence of Garbor-like filters, reminiscent of responses in primary visual cortex. In the last line of work, we model the state of a cortical excitatory-inhibitory network during complex adaptive stimuli. Using a rate model with Wilson-Cowan dynamics, we demonstrate that simple non-linearities in the signal transferred from inhibitory to excitatory neurons can account for real neural recordings taken from auditory cortex. This work establishes and tests

  20. Demixed principal component analysis of neural population data.

    Science.gov (United States)

    Kobak, Dmitry; Brendel, Wieland; Constantinidis, Christos; Feierstein, Claudia E; Kepecs, Adam; Mainen, Zachary F; Qi, Xue-Lian; Romo, Ranulfo; Uchida, Naoshige; Machens, Christian K

    2016-04-12

    Neurons in higher cortical areas, such as the prefrontal cortex, are often tuned to a variety of sensory and motor variables, and are therefore said to display mixed selectivity. This complexity of single neuron responses can obscure what information these areas represent and how it is represented. Here we demonstrate the advantages of a new dimensionality reduction technique, demixed principal component analysis (dPCA), that decomposes population activity into a few components. In addition to systematically capturing the majority of the variance of the data, dPCA also exposes the dependence of the neural representation on task parameters such as stimuli, decisions, or rewards. To illustrate our method we reanalyze population data from four datasets comprising different species, different cortical areas and different experimental tasks. In each case, dPCA provides a concise way of visualizing the data that summarizes the task-dependent features of the population response in a single figure.

  1. Synovial CD4+ T-cell-derived GM-CSF supports the differentiation of an inflammatory dendritic cell population in rheumatoid arthritis

    Science.gov (United States)

    Reynolds, G; Gibbon, J R; Pratt, A G; Wood, M J; Coady, D; Raftery, G; Lorenzi, A R; Gray, A; Filer, A; Buckley, C D; Haniffa, M A; Isaacs, J D; Hilkens, C M U

    2016-01-01

    Objective A population of synovial inflammatory dendritic cells (infDCs) has recently been identified in rheumatoid arthritis (RA) and is thought to be monocyte-derived. Here, we investigated the role and source of granulocyte macrophage-colony-stimulating factor (GM-CSF) in the differentiation of synovial infDC in RA. Methods Production of GM-CSF by peripheral blood (PB) and synovial fluid (SF) CD4+ T cells was assessed by ELISA and flow cytometry. In vitro CD4+ T-cell polarisation experiments were performed with T-cell activating CD2/CD3/CD28-coated beads in the absence or presence of pro-Th1 or pro-Th17 cytokines. CD1c+ DC and CD16+ macrophage subsets were flow-sorted and analysed morphologically and functionally (T-cell stimulatory/polarising capacity). Results RA-SF CD4+ T cells produced abundant GM-CSF upon stimulation and significantly more than RA-SF mononuclear cells depleted of CD4+ T cells. GM-CSF-producing T cells were significantly increased in RA-SF compared with non-RA inflammatory arthritis SF, active RA PB and healthy donor PB. GM-CSF-producing CD4+ T cells were expanded by Th1-promoting but not Th17-promoting conditions. Following coculture with RA-SF CD4+ T cells, but not healthy donor PB CD4+ T cells, a subpopulation of monocytes differentiated into CD1c+ infDC; a process dependent on GM-CSF. These infDC displayed potent alloproliferative capacity and enhanced GM-CSF, interleukin-17 and interferon-γ production by CD4+ T cells. InfDC with an identical phenotype to in vitro generated cells were significantly enriched in RA-SF compared with non-RA-SF/tissue/PB. Conclusions We demonstrate a therapeutically tractable feedback loop of GM-CSF secreted by RA synovial CD4+ T cells promoting the differentiation of infDC with potent capacity to induce GM-CSF-producing CD4+ T cells. PMID:25923217

  2. Locally optimal extracellular stimulation for chaotic desynchronization of neural populations.

    Science.gov (United States)

    Wilson, Dan; Moehlis, Jeff

    2014-10-01

    We use optimal control theory to design a methodology to find locally optimal stimuli for desynchronization of a model of neurons with extracellular stimulation. This methodology yields stimuli which lead to positive Lyapunov exponents, and hence desynchronizes a neural population. We analyze this methodology in the presence of interneuron coupling to make predictions about the strength of stimulation required to overcome synchronizing effects of coupling. This methodology suggests a powerful alternative to pulsatile stimuli for deep brain stimulation as it uses less energy than pulsatile stimuli, and could eliminate the time consuming tuning process.

  3. Super-linear Precision in Simple Neural Population Codes

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    Schwab, David; Fiete, Ila

    2015-03-01

    A widely used tool for quantifying the precision with which a population of noisy sensory neurons encodes the value of an external stimulus is the Fisher Information (FI). Maximizing the FI is also a commonly used objective for constructing optimal neural codes. The primary utility and importance of the FI arises because it gives, through the Cramer-Rao bound, the smallest mean-squared error achievable by any unbiased stimulus estimator. However, it is well-known that when neural firing is sparse, optimizing the FI can result in codes that perform very poorly when considering the resulting mean-squared error, a measure with direct biological relevance. Here we construct optimal population codes by minimizing mean-squared error directly and study the scaling properties of the resulting network, focusing on the optimal tuning curve width. We then extend our results to continuous attractor networks that maintain short-term memory of external stimuli in their dynamics. Here we find similar scaling properties in the structure of the interactions that minimize diffusive information loss.

  4. Degraded attentional modulation of cortical neural populations in strabismic amblyopia.

    Science.gov (United States)

    Hou, Chuan; Kim, Yee-Joon; Lai, Xin Jie; Verghese, Preeti

    2016-01-01

    Behavioral studies have reported reduced spatial attention in amblyopia, a developmental disorder of spatial vision. However, the neural populations in the visual cortex linked with these behavioral spatial attention deficits have not been identified. Here, we use functional MRI-informed electroencephalography source imaging to measure the effect of attention on neural population activity in the visual cortex of human adult strabismic amblyopes who were stereoblind. We show that compared with controls, the modulatory effects of selective visual attention on the input from the amblyopic eye are substantially reduced in the primary visual cortex (V1) as well as in extrastriate visual areas hV4 and hMT+. Degraded attentional modulation is also found in the normal-acuity fellow eye in areas hV4 and hMT+ but not in V1. These results provide electrophysiological evidence that abnormal binocular input during a developmental critical period may impact cortical connections between the visual cortex and higher level cortices beyond the known amblyopic losses in V1 and V2, suggesting that a deficit of attentional modulation in the visual cortex is an important component of the functional impairment in amblyopia. Furthermore, we find that degraded attentional modulation in V1 is correlated with the magnitude of interocular suppression and the depth of amblyopia. These results support the view that the visual suppression often seen in strabismic amblyopia might be a form of attentional neglect of the visual input to the amblyopic eye.

  5. Dual roles for spike signaling in cortical neural populations

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    Dana eBallard

    2011-06-01

    Full Text Available A prominent feature of signaling in cortical neurons is that of randomness in the action potential. The output of a typical pyramidal cell can be well fit with a Poisson model, and variations in the Poisson rate repeatedly have been shown to be correlated with stimuli. However while the rate provides a very useful characterization of neural spike data, it may not be the most fundamental description of the signaling code. Recent data showing γ frequency range multi-cell action potential correlations, together with spike timing dependent plasticity, are spurring a re-examination of the classical model, since precise timing codes imply that the generation of spikes is essentially deterministic. Could the observed Poisson randomness and timing determinism reflect two separate modes of communication, or do they somehow derive from a single process? We investigate in a timing-based model whether the apparent incompatibility between these probabilistic and deterministic observations may be resolved by examining how spikes could be used in the underlying neural circuits. The crucial component of this model draws on dual roles for spike signaling. In learning receptive fields from ensembles of inputs, spikes need to behave probabilistically, whereas for fast signaling of individual stimuli, the spikes need to behave deterministically. Our simulations show that this combination is possible if deterministic signals using γ latency coding are probabilistically routed through different members of a cortical cell population at different times. This model exhibits standard features characteristic of Poisson models such as orientation tuning post-stimulus histograms and exponential interval histograms. In addition it makes testable predictions that follow from the γ latency coding.

  6. Inhibition of Notch Signaling in Human Embryonic Stem Cell-Derived Neural Stem Cells Delays G1/S Phase Transition and Accelerates Neuronal Differentiation In Vitro and In Vivo

    Czech Academy of Sciences Publication Activity Database

    Borghese, L.; Doležalová, Dáša; Opitz, T.; Haupt, S.; Leinhaas, A.; Steinfarz, B.; Koch, P.; Edenhofer, F.; Hampl, Aleš; Brüstle, O.

    2010-01-01

    Roč. 28, č. 5 (2010), s. 955-964 ISSN 1066-5099 Grant - others:GA MŠk(CZ) 1M0538; EC FP6 project ESTOOLS(XE) LSHG-CT-2006-018739; EC FP7 project NeuroStemcell(XE) HEALTH-2007-B-22943 Program:1M Institutional research plan: CEZ:AV0Z50390703 Keywords : neural stem cells * notch * neuron Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 7.871, year: 2010

  7. Location coding by opponent neural populations in the auditory cortex.

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    G Christopher Stecker

    2005-03-01

    Full Text Available Although the auditory cortex plays a necessary role in sound localization, physiological investigations in the cortex reveal inhomogeneous sampling of auditory space that is difficult to reconcile with localization behavior under the assumption of local spatial coding. Most neurons respond maximally to sounds located far to the left or right side, with few neurons tuned to the frontal midline. Paradoxically, psychophysical studies show optimal spatial acuity across the frontal midline. In this paper, we revisit the problem of inhomogeneous spatial sampling in three fields of cat auditory cortex. In each field, we confirm that neural responses tend to be greatest for lateral positions, but show the greatest modulation for near-midline source locations. Moreover, identification of source locations based on cortical responses shows sharp discrimination of left from right but relatively inaccurate discrimination of locations within each half of space. Motivated by these findings, we explore an opponent-process theory in which sound-source locations are represented by differences in the activity of two broadly tuned channels formed by contra- and ipsilaterally preferring neurons. Finally, we demonstrate a simple model, based on spike-count differences across cortical populations, that provides bias-free, level-invariant localization-and thus also a solution to the "binding problem" of associating spatial information with other nonspatial attributes of sounds.

  8. Small-scale screening of anticancer drugs acting specifically on neural stem/progenitor cells derived from human-induced pluripotent stem cells using a time-course cytotoxicity test

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    Hayato Fukusumi

    2018-01-01

    Full Text Available Since the development of human-induced pluripotent stem cells (hiPSCs, various types of hiPSC-derived cells have been established for regenerative medicine and drug development. Neural stem/progenitor cells (NSPCs derived from hiPSCs (hiPSC-NSPCs have shown benefits for regenerative therapy of the central nervous system. However, owing to their intrinsic proliferative potential, therapies using transplanted hiPSC-NSPCs carry an inherent risk of undesired growth in vivo. Therefore, it is important to find cytotoxic drugs that can specifically target overproliferative transplanted hiPSC-NSPCs without damaging the intrinsic in vivo stem-cell system. Here, we examined the chemosensitivity of hiPSC-NSPCs and human neural tissue—derived NSPCs (hN-NSPCs to the general anticancer drugs cisplatin, etoposide, mercaptopurine, and methotrexate. A time-course analysis of neurospheres in a microsphere array identified cisplatin and etoposide as fast-acting drugs, and mercaptopurine and methotrexate as slow-acting drugs. Notably, the slow-acting drugs were eventually cytotoxic to hiPSC-NSPCs but not to hN-NSPCs, a phenomenon not evident in the conventional endpoint assay on day 2 of treatment. Our results indicate that slow-acting drugs can distinguish hiPSC-NSPCs from hN-NSPCs and may provide an effective backup safety measure in stem-cell transplant therapies.

  9. Small-scale screening of anticancer drugs acting specifically on neural stem/progenitor cells derived from human-induced pluripotent stem cells using a time-course cytotoxicity test.

    Science.gov (United States)

    Fukusumi, Hayato; Handa, Yukako; Shofuda, Tomoko; Kanemura, Yonehiro

    2018-01-01

    Since the development of human-induced pluripotent stem cells (hiPSCs), various types of hiPSC-derived cells have been established for regenerative medicine and drug development. Neural stem/progenitor cells (NSPCs) derived from hiPSCs (hiPSC-NSPCs) have shown benefits for regenerative therapy of the central nervous system. However, owing to their intrinsic proliferative potential, therapies using transplanted hiPSC-NSPCs carry an inherent risk of undesired growth in vivo . Therefore, it is important to find cytotoxic drugs that can specifically target overproliferative transplanted hiPSC-NSPCs without damaging the intrinsic in vivo stem-cell system. Here, we examined the chemosensitivity of hiPSC-NSPCs and human neural tissue-derived NSPCs (hN-NSPCs) to the general anticancer drugs cisplatin, etoposide, mercaptopurine, and methotrexate. A time-course analysis of neurospheres in a microsphere array identified cisplatin and etoposide as fast-acting drugs, and mercaptopurine and methotrexate as slow-acting drugs. Notably, the slow-acting drugs were eventually cytotoxic to hiPSC-NSPCs but not to hN-NSPCs, a phenomenon not evident in the conventional endpoint assay on day 2 of treatment. Our results indicate that slow-acting drugs can distinguish hiPSC-NSPCs from hN-NSPCs and may provide an effective backup safety measure in stem-cell transplant therapies.

  10. Event-driven simulation of neural population synchronization facilitated by electrical coupling.

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    Carrillo, Richard R; Ros, Eduardo; Barbour, Boris; Boucheny, Christian; Coenen, Olivier

    2007-02-01

    Most neural communication and processing tasks are driven by spikes. This has enabled the application of the event-driven simulation schemes. However the simulation of spiking neural networks based on complex models that cannot be simplified to analytical expressions (requiring numerical calculation) is very time consuming. Here we describe briefly an event-driven simulation scheme that uses pre-calculated table-based neuron characterizations to avoid numerical calculations during a network simulation, allowing the simulation of large-scale neural systems. More concretely we explain how electrical coupling can be simulated efficiently within this computation scheme, reproducing synchronization processes observed in detailed simulations of neural populations.

  11. Dynamical networks: Finding, measuring, and tracking neural population activity using network science

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    Mark D. Humphries

    2017-12-01

    Full Text Available Systems neuroscience is in a headlong rush to record from as many neurons at the same time as possible. As the brain computes and codes using neuron populations, it is hoped these data will uncover the fundamentals of neural computation. But with hundreds, thousands, or more simultaneously recorded neurons come the inescapable problems of visualizing, describing, and quantifying their interactions. Here I argue that network science provides a set of scalable, analytical tools that already solve these problems. By treating neurons as nodes and their interactions as links, a single network can visualize and describe an arbitrarily large recording. I show that with this description we can quantify the effects of manipulating a neural circuit, track changes in population dynamics over time, and quantitatively define theoretical concepts of neural populations such as cell assemblies. Using network science as a core part of analyzing population recordings will thus provide both qualitative and quantitative advances to our understanding of neural computation.

  12. Knowledge extraction from evolving spiking neural networks with rank order population coding.

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    Soltic, Snjezana; Kasabov, Nikola

    2010-12-01

    This paper demonstrates how knowledge can be extracted from evolving spiking neural networks with rank order population coding. Knowledge discovery is a very important feature of intelligent systems. Yet, a disproportionally small amount of research is centered on the issue of knowledge extraction from spiking neural networks which are considered to be the third generation of artificial neural networks. The lack of knowledge representation compatibility is becoming a major detriment to end users of these networks. We show that a high-level knowledge can be obtained from evolving spiking neural networks. More specifically, we propose a method for fuzzy rule extraction from an evolving spiking network with rank order population coding. The proposed method was used for knowledge discovery on two benchmark taste recognition problems where the knowledge learnt by an evolving spiking neural network was extracted in the form of zero-order Takagi-Sugeno fuzzy IF-THEN rules.

  13. Genetic and Chemical Correction of Cholesterol Accumulation and Impaired Autophagy in Hepatic and Neural Cells Derived from Niemann-Pick Type C Patient-Specific iPS Cells

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    Dorothea Maetzel

    2014-06-01

    Full Text Available Niemann-Pick type C (NPC disease is a fatal inherited lipid storage disorder causing severe neurodegeneration and liver dysfunction with only limited treatment options for patients. Loss of NPC1 function causes defects in cholesterol metabolism and has recently been implicated in deregulation of autophagy. Here, we report the generation of isogenic pairs of NPC patient-specific induced pluripotent stem cells (iPSCs using transcription activator-like effector nucleases (TALENs. We observed decreased cell viability, cholesterol accumulation, and dysfunctional autophagic flux in NPC1-deficient human hepatic and neural cells. Genetic correction of a disease-causing mutation rescued these defects and directly linked NPC1 protein function to impaired cholesterol metabolism and autophagy. Screening for autophagy-inducing compounds in disease-affected human cells showed cell type specificity. Carbamazepine was found to be cytoprotective and effective in restoring the autophagy defects in both NPC1-deficient hepatic and neuronal cells and therefore may be a promising treatment option with overall benefit for NPC disease.

  14. Targeted Inhibition of the miR-199a/214 Cluster by CRISPR Interference Augments the Tumor Tropism of Human Induced Pluripotent Stem Cell-Derived Neural Stem Cells under Hypoxic Condition

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    Yumei Luo

    2016-01-01

    Full Text Available The human induced pluripotent stem cell (hiPSC provides a breakthrough approach that helps overcoming ethical and allergenic challenges posed in application of neural stem cells (NSCs in targeted cancer gene therapy. However, the tumor-tropic capacity of hiPSC-derived NSCs (hiPS-NSCs still has much room to improve. Here we attempted to promote the tumor tropism of hiPS-NSCs by manipulating the activity of endogenous miR-199a/214 cluster that is involved in regulation of hypoxia-stimulated cell migration. We first developed a baculovirus-delivered CRISPR interference (CRISPRi system that sterically blocked the E-box element in the promoter of the miR-199a/214 cluster with an RNA-guided catalytically dead Cas9 (dCas9. We then applied this CRISPRi system to hiPS-NSCs and successfully suppressed the expression of miR-199a-5p, miR-199a-3p, and miR-214 in the microRNA gene cluster. Meanwhile, the expression levels of their targets related to regulation of hypoxia-stimulated cell migration, such as HIF1A, MET, and MAPK1, were upregulated. Further migration assays demonstrated that the targeted inhibition of the miR-199a/214 cluster significantly enhanced the tumor tropism of hiPS-NSCs both in vitro and in vivo. These findings suggest a novel application of CRISPRi in NSC-based tumor-targeted gene therapy.

  15. Towards a theory of cortical columns: From spiking neurons to interacting neural populations of finite size

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    Gerstner, Wulfram

    2017-01-01

    Neural population equations such as neural mass or field models are widely used to study brain activity on a large scale. However, the relation of these models to the properties of single neurons is unclear. Here we derive an equation for several interacting populations at the mesoscopic scale starting from a microscopic model of randomly connected generalized integrate-and-fire neuron models. Each population consists of 50–2000 neurons of the same type but different populations account for different neuron types. The stochastic population equations that we find reveal how spike-history effects in single-neuron dynamics such as refractoriness and adaptation interact with finite-size fluctuations on the population level. Efficient integration of the stochastic mesoscopic equations reproduces the statistical behavior of the population activities obtained from microscopic simulations of a full spiking neural network model. The theory describes nonlinear emergent dynamics such as finite-size-induced stochastic transitions in multistable networks and synchronization in balanced networks of excitatory and inhibitory neurons. The mesoscopic equations are employed to rapidly integrate a model of a cortical microcircuit consisting of eight neuron types, which allows us to predict spontaneous population activities as well as evoked responses to thalamic input. Our theory establishes a general framework for modeling finite-size neural population dynamics based on single cell and synapse parameters and offers an efficient approach to analyzing cortical circuits and computations. PMID:28422957

  16. Towards a theory of cortical columns: From spiking neurons to interacting neural populations of finite size.

    Science.gov (United States)

    Schwalger, Tilo; Deger, Moritz; Gerstner, Wulfram

    2017-04-01

    Neural population equations such as neural mass or field models are widely used to study brain activity on a large scale. However, the relation of these models to the properties of single neurons is unclear. Here we derive an equation for several interacting populations at the mesoscopic scale starting from a microscopic model of randomly connected generalized integrate-and-fire neuron models. Each population consists of 50-2000 neurons of the same type but different populations account for different neuron types. The stochastic population equations that we find reveal how spike-history effects in single-neuron dynamics such as refractoriness and adaptation interact with finite-size fluctuations on the population level. Efficient integration of the stochastic mesoscopic equations reproduces the statistical behavior of the population activities obtained from microscopic simulations of a full spiking neural network model. The theory describes nonlinear emergent dynamics such as finite-size-induced stochastic transitions in multistable networks and synchronization in balanced networks of excitatory and inhibitory neurons. The mesoscopic equations are employed to rapidly integrate a model of a cortical microcircuit consisting of eight neuron types, which allows us to predict spontaneous population activities as well as evoked responses to thalamic input. Our theory establishes a general framework for modeling finite-size neural population dynamics based on single cell and synapse parameters and offers an efficient approach to analyzing cortical circuits and computations.

  17. Bilingualism provides a neural reserve for aging populations.

    Science.gov (United States)

    Abutalebi, Jubin; Guidi, Lucia; Borsa, Virginia; Canini, Matteo; Della Rosa, Pasquale A; Parris, Ben A; Weekes, Brendan S

    2015-03-01

    It has been postulated that bilingualism may act as a cognitive reserve and recent behavioral evidence shows that bilinguals are diagnosed with dementia about 4-5 years later compared to monolinguals. In the present study, we investigated the neural basis of these putative protective effects in a group of aging bilinguals as compared to a matched monolingual control group. For this purpose, participants completed the Erikson Flanker task and their performance was correlated to gray matter (GM) volume in order to investigate if cognitive performance predicts GM volume specifically in areas affected by aging. We performed an ex-Gaussian analysis on the resulting RTs and report that aging bilinguals performed better than aging monolinguals on the Flanker task. Bilingualism was overall associated with increased GM in the ACC. Likewise, aging induced effects upon performance correlated only for monolinguals to decreased gray matter in the DLPFC. Taken together, these neural regions might underlie the benefits of bilingualism and act as a neural reserve that protects against the cognitive decline that occurs during aging. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. The role of stochasticity in an information-optimal neural population code

    International Nuclear Information System (INIS)

    Stocks, N G; Nikitin, A P; McDonnell, M D; Morse, R P

    2009-01-01

    In this paper we consider the optimisation of Shannon mutual information (MI) in the context of two model neural systems. The first is a stochastic pooling network (population) of McCulloch-Pitts (MP) type neurons (logical threshold units) subject to stochastic forcing; the second is (in a rate coding paradigm) a population of neurons that each displays Poisson statistics (the so called 'Poisson neuron'). The mutual information is optimised as a function of a parameter that characterises the 'noise level'-in the MP array this parameter is the standard deviation of the noise; in the population of Poisson neurons it is the window length used to determine the spike count. In both systems we find that the emergent neural architecture and, hence, code that maximises the MI is strongly influenced by the noise level. Low noise levels leads to a heterogeneous distribution of neural parameters (diversity), whereas, medium to high noise levels result in the clustering of neural parameters into distinct groups that can be interpreted as subpopulations. In both cases the number of subpopulations increases with a decrease in noise level. Our results suggest that subpopulations are a generic feature of an information optimal neural population.

  19. The role of stochasticity in an information-optimal neural population code

    Energy Technology Data Exchange (ETDEWEB)

    Stocks, N G; Nikitin, A P [School of Engineering, University of Warwick, Coventry CV4 7AL (United Kingdom); McDonnell, M D [Institute for Telecommunications Research, University of South Australia, SA 5095 (Australia); Morse, R P, E-mail: n.g.stocks@warwick.ac.u [School of Life and Health Sciences, Aston University, Birmingham B4 7ET (United Kingdom)

    2009-12-01

    In this paper we consider the optimisation of Shannon mutual information (MI) in the context of two model neural systems. The first is a stochastic pooling network (population) of McCulloch-Pitts (MP) type neurons (logical threshold units) subject to stochastic forcing; the second is (in a rate coding paradigm) a population of neurons that each displays Poisson statistics (the so called 'Poisson neuron'). The mutual information is optimised as a function of a parameter that characterises the 'noise level'-in the MP array this parameter is the standard deviation of the noise; in the population of Poisson neurons it is the window length used to determine the spike count. In both systems we find that the emergent neural architecture and, hence, code that maximises the MI is strongly influenced by the noise level. Low noise levels leads to a heterogeneous distribution of neural parameters (diversity), whereas, medium to high noise levels result in the clustering of neural parameters into distinct groups that can be interpreted as subpopulations. In both cases the number of subpopulations increases with a decrease in noise level. Our results suggest that subpopulations are a generic feature of an information optimal neural population.

  20. Neural population-level memory traces in the mouse hippocampus.

    Science.gov (United States)

    Chen, Guifen; Wang, L Phillip; Tsien, Joe Z

    2009-12-16

    One of the fundamental goals in neurosciences is to elucidate the formation and retrieval of brain's associative memory traces in real-time. Here, we describe real-time neural ensemble transient dynamics in the mouse hippocampal CA1 region and demonstrate their relationships with behavioral performances during both learning and recall. We employed the classic trace fear conditioning paradigm involving a neutral tone followed by a mild foot-shock 20 seconds later. Our large-scale recording and decoding methods revealed that conditioned tone responses and tone-shock association patterns were not present in CA1 during the first pairing, but emerged quickly after multiple pairings. These encoding patterns showed increased immediate-replay, correlating tightly with increased immediate-freezing during learning. Moreover, during contextual recall, these patterns reappeared in tandem six-to-fourteen times per minute, again correlating tightly with behavioral recall. Upon traced tone recall, while various fear memories were retrieved, the shock traces exhibited a unique recall-peak around the 20-second trace interval, further signifying the memory of time for the expected shock. Therefore, our study has revealed various real-time associative memory traces during learning and recall in CA1, and demonstrates that real-time memory traces can be decoded on a moment-to-moment basis over any single trial.

  1. Neural population-level memory traces in the mouse hippocampus.

    Directory of Open Access Journals (Sweden)

    Guifen Chen

    2009-12-01

    Full Text Available One of the fundamental goals in neurosciences is to elucidate the formation and retrieval of brain's associative memory traces in real-time. Here, we describe real-time neural ensemble transient dynamics in the mouse hippocampal CA1 region and demonstrate their relationships with behavioral performances during both learning and recall. We employed the classic trace fear conditioning paradigm involving a neutral tone followed by a mild foot-shock 20 seconds later. Our large-scale recording and decoding methods revealed that conditioned tone responses and tone-shock association patterns were not present in CA1 during the first pairing, but emerged quickly after multiple pairings. These encoding patterns showed increased immediate-replay, correlating tightly with increased immediate-freezing during learning. Moreover, during contextual recall, these patterns reappeared in tandem six-to-fourteen times per minute, again correlating tightly with behavioral recall. Upon traced tone recall, while various fear memories were retrieved, the shock traces exhibited a unique recall-peak around the 20-second trace interval, further signifying the memory of time for the expected shock. Therefore, our study has revealed various real-time associative memory traces during learning and recall in CA1, and demonstrates that real-time memory traces can be decoded on a moment-to-moment basis over any single trial.

  2. Probabilistic models for neural populations that naturally capture global coupling and criticality.

    Science.gov (United States)

    Humplik, Jan; Tkačik, Gašper

    2017-09-01

    Advances in multi-unit recordings pave the way for statistical modeling of activity patterns in large neural populations. Recent studies have shown that the summed activity of all neurons strongly shapes the population response. A separate recent finding has been that neural populations also exhibit criticality, an anomalously large dynamic range for the probabilities of different population activity patterns. Motivated by these two observations, we introduce a class of probabilistic models which takes into account the prior knowledge that the neural population could be globally coupled and close to critical. These models consist of an energy function which parametrizes interactions between small groups of neurons, and an arbitrary positive, strictly increasing, and twice differentiable function which maps the energy of a population pattern to its probability. We show that: 1) augmenting a pairwise Ising model with a nonlinearity yields an accurate description of the activity of retinal ganglion cells which outperforms previous models based on the summed activity of neurons; 2) prior knowledge that the population is critical translates to prior expectations about the shape of the nonlinearity; 3) the nonlinearity admits an interpretation in terms of a continuous latent variable globally coupling the system whose distribution we can infer from data. Our method is independent of the underlying system's state space; hence, it can be applied to other systems such as natural scenes or amino acid sequences of proteins which are also known to exhibit criticality.

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

    Science.gov (United States)

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

    2016-01-01

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

  4. Population equations for degree-heterogenous neural networks

    Science.gov (United States)

    Kähne, M.; Sokolov, I. M.; Rüdiger, S.

    2017-11-01

    We develop a statistical framework for studying recurrent networks with broad distributions of the number of synaptic links per neuron. We treat each group of neurons with equal input degree as one population and derive a system of equations determining the population-averaged firing rates. The derivation rests on an assumption of a large number of neurons and, additionally, an assumption of a large number of synapses per neuron. For the case of binary neurons, analytical solutions can be constructed, which correspond to steps in the activity versus degree space. We apply this theory to networks with degree-correlated topology and show that complex, multi-stable regimes can result for increasing correlations. Our work is motivated by the recent finding of subnetworks of highly active neurons and the fact that these neurons tend to be connected to each other with higher probability.

  5. Neural signatures of attention: insights from decoding population activity patterns.

    Science.gov (United States)

    Sapountzis, Panagiotis; Gregoriou, Georgia G

    2018-01-01

    Understanding brain function and the computations that individual neurons and neuronal ensembles carry out during cognitive functions is one of the biggest challenges in neuroscientific research. To this end, invasive electrophysiological studies have provided important insights by recording the activity of single neurons in behaving animals. To average out noise, responses are typically averaged across repetitions and across neurons that are usually recorded on different days. However, the brain makes decisions on short time scales based on limited exposure to sensory stimulation by interpreting responses of populations of neurons on a moment to moment basis. Recent studies have employed machine-learning algorithms in attention and other cognitive tasks to decode the information content of distributed activity patterns across neuronal ensembles on a single trial basis. Here, we review results from studies that have used pattern-classification decoding approaches to explore the population representation of cognitive functions. These studies have offered significant insights into population coding mechanisms. Moreover, we discuss how such advances can aid the development of cognitive brain-computer interfaces.

  6. Analysis of Neural Stem Cells from Human Cortical Brain Structures In Vitro.

    Science.gov (United States)

    Aleksandrova, M A; Poltavtseva, R A; Marei, M V; Sukhikh, G T

    2016-05-01

    Comparative immunohistochemical analysis of the neocortex from human fetuses showed that neural stem and progenitor cells are present in the brain throughout the gestation period, at least from week 8 through 26. At the same time, neural stem cells from the first and second trimester fetuses differed by the distribution, morphology, growth, and quantity. Immunocytochemical analysis of neural stem cells derived from fetuses at different gestation terms and cultured under different conditions showed their differentiation capacity. Detailed analysis of neural stem cell populations derived from fetuses on gestation weeks 8-9, 18-20, and 26 expressing Lex/SSEA1 was performed.

  7. Neural Population Dynamics during Reaching Are Better Explained by a Dynamical System than Representational Tuning.

    Science.gov (United States)

    Michaels, Jonathan A; Dann, Benjamin; Scherberger, Hansjörg

    2016-11-01

    Recent models of movement generation in motor cortex have sought to explain neural activity not as a function of movement parameters, known as representational models, but as a dynamical system acting at the level of the population. Despite evidence supporting this framework, the evaluation of representational models and their integration with dynamical systems is incomplete in the literature. Using a representational velocity-tuning based simulation of center-out reaching, we show that incorporating variable latency offsets between neural activity and kinematics is sufficient to generate rotational dynamics at the level of neural populations, a phenomenon observed in motor cortex. However, we developed a covariance-matched permutation test (CMPT) that reassigns neural data between task conditions independently for each neuron while maintaining overall neuron-to-neuron relationships, revealing that rotations based on the representational model did not uniquely depend on the underlying condition structure. In contrast, rotations based on either a dynamical model or motor cortex data depend on this relationship, providing evidence that the dynamical model more readily explains motor cortex activity. Importantly, implementing a recurrent neural network we demonstrate that both representational tuning properties and rotational dynamics emerge, providing evidence that a dynamical system can reproduce previous findings of representational tuning. Finally, using motor cortex data in combination with the CMPT, we show that results based on small numbers of neurons or conditions should be interpreted cautiously, potentially informing future experimental design. Together, our findings reinforce the view that representational models lack the explanatory power to describe complex aspects of single neuron and population level activity.

  8. Neural coding in the visual system of Drosophila melanogaster: How do small neural populations support visually guided behaviours?

    Science.gov (United States)

    Dewar, Alex D M; Wystrach, Antoine; Philippides, Andrew; Graham, Paul

    2017-10-01

    All organisms wishing to survive and reproduce must be able to respond adaptively to a complex, changing world. Yet the computational power available is constrained by biology and evolution, favouring mechanisms that are parsimonious yet robust. Here we investigate the information carried in small populations of visually responsive neurons in Drosophila melanogaster. These so-called 'ring neurons', projecting to the ellipsoid body of the central complex, are reported to be necessary for complex visual tasks such as pattern recognition and visual navigation. Recently the receptive fields of these neurons have been mapped, allowing us to investigate how well they can support such behaviours. For instance, in a simulation of classic pattern discrimination experiments, we show that the pattern of output from the ring neurons matches observed fly behaviour. However, performance of the neurons (as with flies) is not perfect and can be easily improved with the addition of extra neurons, suggesting the neurons' receptive fields are not optimised for recognising abstract shapes, a conclusion which casts doubt on cognitive explanations of fly behaviour in pattern recognition assays. Using artificial neural networks, we then assess how easy it is to decode more general information about stimulus shape from the ring neuron population codes. We show that these neurons are well suited for encoding information about size, position and orientation, which are more relevant behavioural parameters for a fly than abstract pattern properties. This leads us to suggest that in order to understand the properties of neural systems, one must consider how perceptual circuits put information at the service of behaviour.

  9. Population coding and decoding in a neural field: a computational study.

    Science.gov (United States)

    Wu, Si; Amari, Shun-Ichi; Nakahara, Hiroyuki

    2002-05-01

    This study uses a neural field model to investigate computational aspects of population coding and decoding when the stimulus is a single variable. A general prototype model for the encoding process is proposed, in which neural responses are correlated, with strength specified by a gaussian function of their difference in preferred stimuli. Based on the model, we study the effect of correlation on the Fisher information, compare the performances of three decoding methods that differ in the amount of encoding information being used, and investigate the implementation of the three methods by using a recurrent network. This study not only rediscovers main results in existing literatures in a unified way, but also reveals important new features, especially when the neural correlation is strong. As the neural correlation of firing becomes larger, the Fisher information decreases drastically. We confirm that as the width of correlation increases, the Fisher information saturates and no longer increases in proportion to the number of neurons. However, we prove that as the width increases further--wider than (sqrt)2 times the effective width of the turning function--the Fisher information increases again, and it increases without limit in proportion to the number of neurons. Furthermore, we clarify the asymptotic efficiency of the maximum likelihood inference (MLI) type of decoding methods for correlated neural signals. It shows that when the correlation covers a nonlocal range of population (excepting the uniform correlation and when the noise is extremely small), the MLI type of method, whose decoding error satisfies the Cauchy-type distribution, is not asymptotically efficient. This implies that the variance is no longer adequate to measure decoding accuracy.

  10. Stochastic Nonlinear Evolutional Model of the Large-Scaled Neuronal Population and Dynamic Neural Coding Subject to Stimulation

    International Nuclear Information System (INIS)

    Wang Rubin; Yu Wei

    2005-01-01

    In this paper, we investigate how the population of neuronal oscillators deals with information and the dynamic evolution of neural coding when the external stimulation acts on it. Numerically computing method is used to describe the evolution process of neural coding in three-dimensioned space. The numerical result proves that only the suitable stimulation can change the coupling structure and plasticity of neurons

  11. Measures of Coupling between Neural Populations Based on Granger Causality Principle.

    Science.gov (United States)

    Kaminski, Maciej; Brzezicka, Aneta; Kaminski, Jan; Blinowska, Katarzyna J

    2016-01-01

    This paper shortly reviews the measures used to estimate neural synchronization in experimental settings. Our focus is on multivariate measures of dependence based on the Granger causality (G-causality) principle, their applications and performance in respect of robustness to noise, volume conduction, common driving, and presence of a "weak node." Application of G-causality measures to EEG, intracranial signals and fMRI time series is addressed. G-causality based measures defined in the frequency domain allow the synchronization between neural populations and the directed propagation of their electrical activity to be determined. The time-varying G-causality based measure Short-time Directed Transfer Function (SDTF) supplies information on the dynamics of synchronization and the organization of neural networks. Inspection of effective connectivity patterns indicates a modular structure of neural networks, with a stronger coupling within modules than between them. The hypothetical plausible mechanism of information processing, suggested by the identified synchronization patterns, is communication between tightly coupled modules intermitted by sparser interactions providing synchronization of distant structures.

  12. Measures of coupling between neural populations based on Granger causality principle

    Directory of Open Access Journals (Sweden)

    Maciej Kaminski

    2016-10-01

    Full Text Available This paper shortly reviews the measures used to estimate neural synchronization in experimental settings. Our focus is on multivariate measures of dependence based on the Granger causality (G-causality principle, their applications and performance in respect of robustness to noise, volume conduction, common driving, and presence of a weak node. Application of G-causality measures to EEG, intracranial signals and fMRI time series is addressed. G-causality based measures defined in the frequency domain allow the synchronization between neural populations and the directed propagation of their electrical activity to be determined. The time-varying G-causality based measure Short-time Directed Transfer Function (SDTF supplies information on the dynamics of synchronization and the organization of neural networks. Inspection of effective connectivity patterns indicates a modular structure of neural networks, with a stronger coupling within modules than between them. The hypothetical plausible mechanism of information processing, suggested by the identified synchronization patterns, is communication between tightly coupled modules intermitted by sparser interactions providing synchronization of distant structures.

  13. Population-wide distributions of neural activity during perceptual decision-making

    Science.gov (United States)

    Machens, Christian

    2018-01-01

    Cortical activity involves large populations of neurons, even when it is limited to functionally coherent areas. Electrophysiological recordings, on the other hand, involve comparatively small neural ensembles, even when modern-day techniques are used. Here we review results which have started to fill the gap between these two scales of inquiry, by shedding light on the statistical distributions of activity in large populations of cells. We put our main focus on data recorded in awake animals that perform simple decision-making tasks and consider statistical distributions of activity throughout cortex, across sensory, associative, and motor areas. We transversally review the complexity of these distributions, from distributions of firing rates and metrics of spike-train structure, through distributions of tuning to stimuli or actions and of choice signals, and finally the dynamical evolution of neural population activity and the distributions of (pairwise) neural interactions. This approach reveals shared patterns of statistical organization across cortex, including: (i) long-tailed distributions of activity, where quasi-silence seems to be the rule for a majority of neurons; that are barely distinguishable between spontaneous and active states; (ii) distributions of tuning parameters for sensory (and motor) variables, which show an extensive extrapolation and fragmentation of their representations in the periphery; and (iii) population-wide dynamics that reveal rotations of internal representations over time, whose traces can be found both in stimulus-driven and internally generated activity. We discuss how these insights are leading us away from the notion of discrete classes of cells, and are acting as powerful constraints on theories and models of cortical organization and population coding. PMID:23123501

  14. A neural population model incorporating dopaminergic neurotransmission during complex voluntary behaviors.

    Directory of Open Access Journals (Sweden)

    Stefan Fürtinger

    2014-11-01

    Full Text Available Assessing brain activity during complex voluntary motor behaviors that require the recruitment of multiple neural sites is a field of active research. Our current knowledge is primarily based on human brain imaging studies that have clear limitations in terms of temporal and spatial resolution. We developed a physiologically informed non-linear multi-compartment stochastic neural model to simulate functional brain activity coupled with neurotransmitter release during complex voluntary behavior, such as speech production. Due to its state-dependent modulation of neural firing, dopaminergic neurotransmission plays a key role in the organization of functional brain circuits controlling speech and language and thus has been incorporated in our neural population model. A rigorous mathematical proof establishing existence and uniqueness of solutions to the proposed model as well as a computationally efficient strategy to numerically approximate these solutions are presented. Simulated brain activity during the resting state and sentence production was analyzed using functional network connectivity, and graph theoretical techniques were employed to highlight differences between the two conditions. We demonstrate that our model successfully reproduces characteristic changes seen in empirical data between the resting state and speech production, and dopaminergic neurotransmission evokes pronounced changes in modeled functional connectivity by acting on the underlying biological stochastic neural model. Specifically, model and data networks in both speech and rest conditions share task-specific network features: both the simulated and empirical functional connectivity networks show an increase in nodal influence and segregation in speech over the resting state. These commonalities confirm that dopamine is a key neuromodulator of the functional connectome of speech control. Based on reproducible characteristic aspects of empirical data, we suggest a number

  15. Forecasting influenza-like illness dynamics for military populations using neural networks and social media.

    Directory of Open Access Journals (Sweden)

    Svitlana Volkova

    Full Text Available This work is the first to take advantage of recurrent neural networks to predict influenza-like illness (ILI dynamics from various linguistic signals extracted from social media data. Unlike other approaches that rely on timeseries analysis of historical ILI data and the state-of-the-art machine learning models, we build and evaluate the predictive power of neural network architectures based on Long Short Term Memory (LSTMs units capable of nowcasting (predicting in "real-time" and forecasting (predicting the future ILI dynamics in the 2011 - 2014 influenza seasons. To build our models we integrate information people post in social media e.g., topics, embeddings, word ngrams, stylistic patterns, and communication behavior using hashtags and mentions. We then quantitatively evaluate the predictive power of different social media signals and contrast the performance of the-state-of-the-art regression models with neural networks using a diverse set of evaluation metrics. Finally, we combine ILI and social media signals to build a joint neural network model for ILI dynamics prediction. Unlike the majority of the existing work, we specifically focus on developing models for local rather than national ILI surveillance, specifically for military rather than general populations in 26 U.S. and six international locations., and analyze how model performance depends on the amount of social media data available per location. Our approach demonstrates several advantages: (a Neural network architectures that rely on LSTM units trained on social media data yield the best performance compared to previously used regression models. (b Previously under-explored language and communication behavior features are more predictive of ILI dynamics than stylistic and topic signals expressed in social media. (c Neural network models learned exclusively from social media signals yield comparable or better performance to the models learned from ILI historical data, thus

  16. Forecasting influenza-like illness dynamics for military populations using neural networks and social media.

    Science.gov (United States)

    Volkova, Svitlana; Ayton, Ellyn; Porterfield, Katherine; Corley, Courtney D

    2017-01-01

    This work is the first to take advantage of recurrent neural networks to predict influenza-like illness (ILI) dynamics from various linguistic signals extracted from social media data. Unlike other approaches that rely on timeseries analysis of historical ILI data and the state-of-the-art machine learning models, we build and evaluate the predictive power of neural network architectures based on Long Short Term Memory (LSTMs) units capable of nowcasting (predicting in "real-time") and forecasting (predicting the future) ILI dynamics in the 2011 - 2014 influenza seasons. To build our models we integrate information people post in social media e.g., topics, embeddings, word ngrams, stylistic patterns, and communication behavior using hashtags and mentions. We then quantitatively evaluate the predictive power of different social media signals and contrast the performance of the-state-of-the-art regression models with neural networks using a diverse set of evaluation metrics. Finally, we combine ILI and social media signals to build a joint neural network model for ILI dynamics prediction. Unlike the majority of the existing work, we specifically focus on developing models for local rather than national ILI surveillance, specifically for military rather than general populations in 26 U.S. and six international locations., and analyze how model performance depends on the amount of social media data available per location. Our approach demonstrates several advantages: (a) Neural network architectures that rely on LSTM units trained on social media data yield the best performance compared to previously used regression models. (b) Previously under-explored language and communication behavior features are more predictive of ILI dynamics than stylistic and topic signals expressed in social media. (c) Neural network models learned exclusively from social media signals yield comparable or better performance to the models learned from ILI historical data, thus, signals from

  17. Study of GABAergic extra-synaptic tonic inhibition in single neurons and neural populations by traversing neural scales: application to propofol-induced anaesthesia.

    Science.gov (United States)

    Hutt, Axel; Buhry, Laure

    2014-12-01

    Anaesthetic agents are known to affect extra-synaptic GABAergic receptors, which induce tonic inhibitory currents. Since these receptors are very sensitive to small concentrations of agents, they are supposed to play an important role in the underlying neural mechanism of general anaesthesia. Moreover anaesthetic agents modulate the encephalographic activity (EEG) of subjects and hence show an effect on neural populations. To understand better the tonic inhibition effect in single neurons on neural populations and hence how it affects the EEG, the work considers single neurons and neural populations in a steady-state and studies numerically and analytically the modulation of their firing rate and nonlinear gain with respect to different levels of tonic inhibition. We consider populations of both type-I (Leaky Integrate-and-Fire model) and type-II (Morris-Lecar model) neurons. To bridge the single neuron description to the population description analytically, a recently proposed statistical approach is employed which allows to derive new analytical expressions for the population firing rate for type-I neurons. In addition, the work shows the derivation of a novel transfer function for type-I neurons as considered in neural mass models and studies briefly the interaction of synaptic and extra-synaptic inhibition. We reveal a strong subtractive and divisive effect of tonic inhibition in type-I neurons, i.e. a shift of the firing rate to higher excitation levels accompanied by a change of the nonlinear gain. Tonic inhibition shortens the excitation window of type-II neurons and their populations while maintaining the nonlinear gain. The gained results are interpreted in the context of recent experimental findings under propofol-induced anaesthesia.

  18. Human Stem Cell Derived Cardiomyocytes: An Alternative ...

    Science.gov (United States)

    Chemical spills and associated deaths in the US has increased 2.6-fold and 16-fold from 1983 to 2012, respectfully. In addition, the number of chemicals to which humans are exposed to in the environment has increased almost 10-fold from 2001 to 2013 within the US. Internationally, a WHO report on the global composite impact of chemicals on health reported that 16% of the total burden of cardiovascular disease was attributed to environmental chemical exposure with 2.5 million deaths per year. Clearly, the cardiovascular system, at all its various developmental and life stages, represents a critical target organ system that can be adversely affected by existing and emerging chemicals (e.g., engineered nanomaterials) in a variety of environmental media. The ability to assess chemical cardiac risk and safety is critically needed but extremely challenging due to the number and categories of chemicals in commerce, as indicated. This presentation\\session will evaluate the use of adult human stem cell derived cardiomyocytes, and existing platforms, as an alternative model to evaluate environmental chemical cardiac toxicity as well as provide key information for the development of predictive adverse outcomes pathways associated with environmental chemical exposures. (This abstract does not represent EPA policy) Rapid and translatable chemical safety screening models for cardiotoxicity current status for informing regulatory decisions, a workshop sponsored by the Society

  19. Increasing magnetite contents of polymeric magnetic particles dramatically improves labeling of neural stem cell transplant populations.

    Science.gov (United States)

    Adams, Christopher F; Rai, Ahmad; Sneddon, Gregor; Yiu, Humphrey H P; Polyak, Boris; Chari, Divya M

    2015-01-01

    Safe and efficient delivery of therapeutic cells to sites of injury/disease in the central nervous system is a key goal for the translation of clinical cell transplantation therapies. Recently, 'magnetic cell localization strategies' have emerged as a promising and safe approach for targeted delivery of magnetic particle (MP) labeled stem cells to pathology sites. For neuroregenerative applications, this approach is limited by the lack of available neurocompatible MPs, and low cell labeling achieved in neural stem/precursor populations. We demonstrate that high magnetite content, self-sedimenting polymeric MPs [unfunctionalized poly(lactic acid) coated, without a transfecting component] achieve efficient labeling (≥90%) of primary neural stem cells (NSCs)-a 'hard-to-label' transplant population of major clinical relevance. Our protocols showed high safety with respect to key stem cell regenerative parameters. Critically, labeled cells were effectively localized in an in vitro flow system by magnetic force highlighting the translational potential of the methods used. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. How fast can we learn maximum entropy models of neural populations?

    Energy Technology Data Exchange (ETDEWEB)

    Ganmor, Elad; Schneidman, Elad [Department of Neuroscience, Weizmann Institute of Science, Rehovot 76100 (Israel); Segev, Ronen, E-mail: elad.ganmor@weizmann.ac.i, E-mail: elad.schneidman@weizmann.ac.i [Department of Life Sciences and Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, Beer-Sheva 84105 (Israel)

    2009-12-01

    Most of our knowledge about how the brain encodes information comes from recordings of single neurons. However, computations in the brain are carried out by large groups of neurons. Modelling the joint activity of many interacting elements is computationally hard because of the large number of possible activity patterns and limited experimental data. Recently it was shown in several different neural systems that maximum entropy pairwise models, which rely only on firing rates and pairwise correlations of neurons, are excellent models for the distribution of activity patterns of neural populations, and in particular, their responses to natural stimuli. Using simultaneous recordings of large groups of neurons in the vertebrate retina responding to naturalistic stimuli, we show here that the relevant statistics required for finding the pairwise model can be accurately estimated within seconds. Furthermore, while higher order statistics may, in theory, improve model accuracy, they are, in practice, harmful for times of up to 20 minutes due to sampling noise. Finally, we demonstrate that trading accuracy for entropy may actually improve model performance when data is limited, and suggest an optimization method that automatically adjusts model constraints in order to achieve good performance.

  1. How fast can we learn maximum entropy models of neural populations?

    International Nuclear Information System (INIS)

    Ganmor, Elad; Schneidman, Elad; Segev, Ronen

    2009-01-01

    Most of our knowledge about how the brain encodes information comes from recordings of single neurons. However, computations in the brain are carried out by large groups of neurons. Modelling the joint activity of many interacting elements is computationally hard because of the large number of possible activity patterns and limited experimental data. Recently it was shown in several different neural systems that maximum entropy pairwise models, which rely only on firing rates and pairwise correlations of neurons, are excellent models for the distribution of activity patterns of neural populations, and in particular, their responses to natural stimuli. Using simultaneous recordings of large groups of neurons in the vertebrate retina responding to naturalistic stimuli, we show here that the relevant statistics required for finding the pairwise model can be accurately estimated within seconds. Furthermore, while higher order statistics may, in theory, improve model accuracy, they are, in practice, harmful for times of up to 20 minutes due to sampling noise. Finally, we demonstrate that trading accuracy for entropy may actually improve model performance when data is limited, and suggest an optimization method that automatically adjusts model constraints in order to achieve good performance.

  2. Generation and purification of human stem cell-derived cardiomyocytes

    NARCIS (Netherlands)

    Schwach, Verena; Passier, Robert

    2016-01-01

    © 2016 International Society of Differentiation Efficient and reproducible generation and purification of human stem cell-derived cardiomyocytes (CMs) is crucial for regenerative medicine, disease modeling, drug screening and study of developmental events during cardiac specification. Established

  3. Artificial neural network models for prediction of cardiovascular autonomic dysfunction in general Chinese population

    Science.gov (United States)

    2013-01-01

    Background The present study aimed to develop an artificial neural network (ANN) based prediction model for cardiovascular autonomic (CA) dysfunction in the general population. Methods We analyzed a previous dataset based on a population sample consisted of 2,092 individuals aged 30–80 years. The prediction models were derived from an exploratory set using ANN analysis. Performances of these prediction models were evaluated in the validation set. Results Univariate analysis indicated that 14 risk factors showed statistically significant association with CA dysfunction (P < 0.05). The mean area under the receiver-operating curve was 0.762 (95% CI 0.732–0.793) for prediction model developed using ANN analysis. The mean sensitivity, specificity, positive and negative predictive values were similar in the prediction models was 0.751, 0.665, 0.330 and 0.924, respectively. All HL statistics were less than 15.0. Conclusion ANN is an effective tool for developing prediction models with high value for predicting CA dysfunction among the general population. PMID:23902963

  4. Shades of grey; Assessing the contribution of the magno- and parvocellular systems to neural processing of the retinal input in the human visual system from the influence of neural population size and its discharge activity on the VEP.

    Science.gov (United States)

    Marcar, Valentine L; Baselgia, Silvana; Lüthi-Eisenegger, Barbara; Jäncke, Lutz

    2018-03-01

    Retinal input processing in the human visual system involves a phasic and tonic neural response. We investigated the role of the magno- and parvocellular systems by comparing the influence of the active neural population size and its discharge activity on the amplitude and latency of four VEP components. We recorded the scalp electric potential of 20 human volunteers viewing a series of dartboard images presented as a pattern reversing and pattern on-/offset stimulus. These patterns were designed to vary both neural population size coding the temporal- and spatial luminance contrast property and the discharge activity of the population involved in a systematic manner. When the VEP amplitude reflected the size of the neural population coding the temporal luminance contrast property of the image, the influence of luminance contrast followed the contrast response function of the parvocellular system. When the VEP amplitude reflected the size of the neural population responding to the spatial luminance contrast property the image, the influence of luminance contrast followed the contrast response function of the magnocellular system. The latencies of the VEP components examined exhibited the same behavior across our stimulus series. This investigation demonstrates the complex interplay of the magno- and parvocellular systems on the neural response as captured by the VEP. It also demonstrates a linear relationship between stimulus property, neural response, and the VEP and reveals the importance of feedback projections in modulating the ongoing neural response. In doing so, it corroborates the conclusions of our previous study.

  5. A computational model incorporating neural stem cell dynamics reproduces glioma incidence across the lifespan in the human population.

    Directory of Open Access Journals (Sweden)

    Roman Bauer

    Full Text Available Glioma is the most common form of primary brain tumor. Demographically, the risk of occurrence increases until old age. Here we present a novel computational model to reproduce the probability of glioma incidence across the lifespan. Previous mathematical models explaining glioma incidence are framed in a rather abstract way, and do not directly relate to empirical findings. To decrease this gap between theory and experimental observations, we incorporate recent data on cellular and molecular factors underlying gliomagenesis. Since evidence implicates the adult neural stem cell as the likely cell-of-origin of glioma, we have incorporated empirically-determined estimates of neural stem cell number, cell division rate, mutation rate and oncogenic potential into our model. We demonstrate that our model yields results which match actual demographic data in the human population. In particular, this model accounts for the observed peak incidence of glioma at approximately 80 years of age, without the need to assert differential susceptibility throughout the population. Overall, our model supports the hypothesis that glioma is caused by randomly-occurring oncogenic mutations within the neural stem cell population. Based on this model, we assess the influence of the (experimentally indicated decrease in the number of neural stem cells and increase of cell division rate during aging. Our model provides multiple testable predictions, and suggests that different temporal sequences of oncogenic mutations can lead to tumorigenesis. Finally, we conclude that four or five oncogenic mutations are sufficient for the formation of glioma.

  6. Structural phenotyping of stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Pasqualini, Francesco Silvio; Sheehy, Sean Paul; Agarwal, Ashutosh; Aratyn-Schaus, Yvonne; Parker, Kevin Kit

    2015-03-10

    Structural phenotyping based on classical image feature detection has been adopted to elucidate the molecular mechanisms behind genetically or pharmacologically induced changes in cell morphology. Here, we developed a set of 11 metrics to capture the increasing sarcomere organization that occurs intracellularly during striated muscle cell development. To test our metrics, we analyzed the localization of the contractile protein α-actinin in a variety of primary and stem-cell derived cardiomyocytes. Further, we combined these metrics with data mining algorithms to unbiasedly score the phenotypic maturity of human-induced pluripotent stem cell-derived cardiomyocytes. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  7. Scaling Properties of Dimensionality Reduction for Neural Populations and Network Models.

    Directory of Open Access Journals (Sweden)

    Ryan C Williamson

    2016-12-01

    Full Text Available Recent studies have applied dimensionality reduction methods to understand how the multi-dimensional structure of neural population activity gives rise to brain function. It is unclear, however, how the results obtained from dimensionality reduction generalize to recordings with larger numbers of neurons and trials or how these results relate to the underlying network structure. We address these questions by applying factor analysis to recordings in the visual cortex of non-human primates and to spiking network models that self-generate irregular activity through a balance of excitation and inhibition. We compared the scaling trends of two key outputs of dimensionality reduction-shared dimensionality and percent shared variance-with neuron and trial count. We found that the scaling properties of networks with non-clustered and clustered connectivity differed, and that the in vivo recordings were more consistent with the clustered network. Furthermore, recordings from tens of neurons were sufficient to identify the dominant modes of shared variability that generalize to larger portions of the network. These findings can help guide the interpretation of dimensionality reduction outputs in regimes of limited neuron and trial sampling and help relate these outputs to the underlying network structure.

  8. Role of SDF1/CXCR4 Interaction in Experimental Hemiplegic Models with Neural Cell Transplantation

    Directory of Open Access Journals (Sweden)

    Noboru Suzuki

    2012-02-01

    Full Text Available Much attention has been focused on neural cell transplantation because of its promising clinical applications. We have reported that embryonic stem (ES cell derived neural stem/progenitor cell transplantation significantly improved motor functions in a hemiplegic mouse model. It is important to understand the molecular mechanisms governing neural regeneration of the damaged motor cortex after the transplantation. Recent investigations disclosed that chemokines participated in the regulation of migration and maturation of neural cell grafts. In this review, we summarize the involvement of inflammatory chemokines including stromal cell derived factor 1 (SDF1 in neural regeneration after ES cell derived neural stem/progenitor cell transplantation in mouse stroke models.

  9. The sign rule and beyond: boundary effects, flexibility, and noise correlations in neural population codes.

    Directory of Open Access Journals (Sweden)

    Yu Hu

    2014-02-01

    Full Text Available Over repeat presentations of the same stimulus, sensory neurons show variable responses. This "noise" is typically correlated between pairs of cells, and a question with rich history in neuroscience is how these noise correlations impact the population's ability to encode the stimulus. Here, we consider a very general setting for population coding, investigating how information varies as a function of noise correlations, with all other aspects of the problem - neural tuning curves, etc. - held fixed. This work yields unifying insights into the role of noise correlations. These are summarized in the form of theorems, and illustrated with numerical examples involving neurons with diverse tuning curves. Our main contributions are as follows. (1 We generalize previous results to prove a sign rule (SR - if noise correlations between pairs of neurons have opposite signs vs. their signal correlations, then coding performance will improve compared to the independent case. This holds for three different metrics of coding performance, and for arbitrary tuning curves and levels of heterogeneity. This generality is true for our other results as well. (2 As also pointed out in the literature, the SR does not provide a necessary condition for good coding. We show that a diverse set of correlation structures can improve coding. Many of these violate the SR, as do experimentally observed correlations. There is structure to this diversity: we prove that the optimal correlation structures must lie on boundaries of the possible set of noise correlations. (3 We provide a novel set of necessary and sufficient conditions, under which the coding performance (in the presence of noise will be as good as it would be if there were no noise present at all.

  10. Stromal cell-derived factor 1α (SDF-1α)

    DEFF Research Database (Denmark)

    Li, Dana; Bjørnager, Louise; Langkilde, Anne

    2016-01-01

    OBJECTIVES: Stromal cell-derived factor 1a (SDF-1α), is a chemokine and is able to home hematopoietic progenitor cells to injured areas of heart tissue for structural repair. Previous studies have found increased levels of SDF-1α in several cardiac diseases, but only few studies have investigated...

  11. Translational applications of adult stem cell-derived organoids

    NARCIS (Netherlands)

    Drost, Jarno; Clevers, Hans

    2017-01-01

    Adult stem cells from a variety of organs can be expanded long-term in vitro as three-dimensional organotypic structures termed organoids. These adult stem cell-derived organoids retain their organ identity and remain genetically stable over long periods of time. The ability to grow organoids from

  12. Forecasting the mortality rates of Indonesian population by using neural network

    Science.gov (United States)

    Safitri, Lutfiani; Mardiyati, Sri; Rahim, Hendrisman

    2018-03-01

    A model that can represent a problem is required in conducting a forecasting. One of the models that has been acknowledged by the actuary community in forecasting mortality rate is the Lee-Certer model. Lee Carter model supported by Neural Network will be used to calculate mortality forecasting in Indonesia. The type of Neural Network used is feedforward neural network aligned with backpropagation algorithm in python programming language. And the final result of this study is mortality rate in forecasting Indonesia for the next few years

  13. Different Angiogenic Potentials of Mesenchymal Stem Cells Derived from Umbilical Artery, Umbilical Vein, and Wharton’s Jelly

    Directory of Open Access Journals (Sweden)

    Lu Xu

    2017-01-01

    Full Text Available Human mesenchymal stem cells derived from the umbilical cord (UC are a favorable source for allogeneic cell therapy. Here, we successfully isolated the stem cells derived from three different compartments of the human UC, including perivascular stem cells derived from umbilical arteries (UCA-PSCs, perivascular stem cells derived from umbilical vein (UCV-PSCs, and mesenchymal stem cells derived from Wharton’s jelly (WJ-MSCs. These cells had the similar phenotype and differentiation potential toward adipocytes, osteoblasts, and neuron-like cells. However, UCA-PSCs and UCV-PSCs had more CD146+ cells than WJ-MSCs (P<0.05. Tube formation assay in vitro showed the largest number of tube-like structures and branch points in UCA-PSCs among the three stem cells. Additionally, the total tube length in UCA-PSCs and UCV-PSCs was significantly longer than in WJ-MSCs (P<0.01. Microarray, qRT-PCR, and Western blot analysis showed that UCA-PSCs had the highest expression of the Notch ligand Jagged1 (JAG1, which is crucial for blood vessel maturation. Knockdown of Jagged1 significantly impaired the angiogenesis in UCA-PSCs. In summary, UCA-PSCs are promising cell populations for clinical use in ischemic diseases.

  14. Rapid Cellular Phenotyping of Human Pluripotent Stem Cell-Derived Cardiomyocytes using a Genetically Encoded Fluorescent Voltage Sensor

    Directory of Open Access Journals (Sweden)

    Jordan S. Leyton-Mange

    2014-02-01

    Full Text Available In addition to their promise in regenerative medicine, pluripotent stem cells have proved to be faithful models of many human diseases. In particular, patient-specific stem cell-derived cardiomyocytes recapitulate key features of several life-threatening cardiac arrhythmia syndromes. For both modeling and regenerative approaches, phenotyping of stem cell-derived tissues is critical. Cellular phenotyping has largely relied upon expression of lineage markers rather than physiologic attributes. This is especially true for cardiomyocytes, in part because electrophysiological recordings are labor intensive. Likewise, most optical voltage indicators suffer from phototoxicity, which damages cells and degrades signal quality. Here we present the use of a genetically encoded fluorescent voltage indicator, ArcLight, which we demonstrate can faithfully report transmembrane potentials in human stem cell-derived cardiomyocytes. We demonstrate the application of this fluorescent sensor in high-throughput, serial phenotyping of differentiating cardiomyocyte populations and in screening for drug-induced cardiotoxicity.

  15. Neural crest stem cell population in craniomaxillofacial development and tissue repair

    Directory of Open Access Journals (Sweden)

    M La Noce

    2014-10-01

    Full Text Available Neural crest cells, delaminating from the neural tube during migration, undergo an epithelial-mesenchymal transition and differentiate into several cell types strongly reinforcing the mesoderm of the craniofacial body area – giving rise to bone, cartilage and other tissues and cells of this human body area. Recent studies on craniomaxillofacial neural crest-derived cells have provided evidence for the tremendous plasticity of these cells. Actually, neural crest cells can respond and adapt to the environment in which they migrate and the cranial mesoderm plays an important role toward patterning the identity of the migrating neural crest cells. In our experience, neural crest-derived stem cells, such as dental pulp stem cells, can actively proliferate, repair bone and give rise to other tissues and cytotypes, including blood vessels, smooth muscle, adipocytes and melanocytes, highlighting that their use in tissue engineering is successful. In this review, we provide an overview of the main pathways involved in neural crest formation, delamination, migration and differentiation; and, in particular, we concentrate our attention on the translatability of the latest scientific progress. Here we try to suggest new ideas and strategies that are needed to fully develop the clinical use of these cells. This effort should involve both researchers/clinicians and improvements in good manufacturing practice procedures. It is important to address studies towards clinical application or take into consideration that studies must have an effective therapeutic prospect for humans. New approaches and ideas must be concentrated also toward stem cell recruitment and activation within the human body, overcoming the classical grafting.

  16. Polygenic risk for five psychiatric disorders and cross-disorder and disorder-specific neural connectivity in two independent populations.

    Science.gov (United States)

    Wang, Tianqi; Zhang, Xiaolong; Li, Ang; Zhu, Meifang; Liu, Shu; Qin, Wen; Li, Jin; Yu, Chunshui; Jiang, Tianzi; Liu, Bing

    2017-01-01

    Major psychiatric disorders, including attention deficit hyperactivity disorder (ADHD), autism (AUT), bipolar disorder (BD), major depressive disorder (MDD), and schizophrenia (SZ), are highly heritable and polygenic. Evidence suggests that these five disorders have both shared and distinct genetic risks and neural connectivity abnormalities. To measure aggregate genetic risks, the polygenic risk score (PGRS) was computed. Two independent general populations (N = 360 and N = 323) were separately examined to investigate whether the cross-disorder PGRS and PGRS for a specific disorder were associated with individual variability in functional connectivity. Consistent altered functional connectivity was found with the bilateral insula: for the left supplementary motor area and the left superior temporal gyrus with the cross-disorder PGRS, for the left insula and right middle and superior temporal lobe associated with the PGRS for autism, for the bilateral midbrain, posterior cingulate, cuneus, and precuneus associated with the PGRS for BD, and for the left angular gyrus and the left dorsolateral prefrontal cortex associated with the PGRS for schizophrenia. No significant functional connectivity was found associated with the PGRS for ADHD and MDD. Our findings indicated that genetic effects on the cross-disorder and disorder-specific neural connectivity of common genetic risk loci are detectable in the general population. Our findings also indicated that polygenic risk contributes to the main neurobiological phenotypes of psychiatric disorders and that identifying cross-disorder and specific functional connectivity related to polygenic risks may elucidate the neural pathways for these disorders.

  17. Circulating cell-derived microparticles in women with pregnancy loss.

    Science.gov (United States)

    Alijotas-Reig, Jaume; Palacio-Garcia, Carles; Farran-Codina, Immaculada; Zarzoso, Cristina; Cabero-Roura, Luis; Vilardell-Tarres, Miquel

    2011-09-01

    To analyze cell-derived microparticles (cMP) in pregnancy loss (PL), both recurrent miscarriages (RM) and unexplained fetal loss (UFL). Non-matched case-control study was performed at Vall d'Hebron Hospital. Cell-derived microparticles of 53 PL cases, 30 with RM, 16 with UFL, and 7 (RM + UFL), were compared to 38 healthy pregnant women. Twenty healthy non-pregnant women act as controls. Cell-derived microparticles were analyzed through flow cytometry. Results are given as total annexin (A5+), endothelial-(CD144+/CD31+ CD41-), platelet-(CD41+), leukocyte-(CD45+) and CD41- c-MP/μL of plasma. Antiphospholipid antibodies (aPLA) were analyzed according to established methods. Comparing PL versus healthy pregnant, we observed a significant endothelial cMP decrease in PL. When comparing RM subgroup with controls, we observed significant decreases in endothelial cMP. When comparing the PL positive for aPLA versus PL-aPLA-negative, no cMP numbering differences were seen. Pregnancy loss seems to be related to endothelial cell activation and/or consumption. A relationship between aPLA and cMP could not be demonstrated. © 2011 John Wiley & Sons A/S.

  18. Attention Effects on Neural Population Representations for Shape and Location Are Stronger in the Ventral than Dorsal Stream

    Science.gov (United States)

    2018-01-01

    Abstract We examined how attention causes neural population representations of shape and location to change in ventral stream (AIT) and dorsal stream (LIP). Monkeys performed two identical delayed-match-to-sample (DMTS) tasks, attending either to shape or location. In AIT, shapes were more discriminable when directing attention to shape rather than location, measured by an increase in mean distance between population response vectors. In LIP, attending to location rather than shape did not increase the discriminability of different stimulus locations. Even when factoring out the change in mean vector response distance, multidimensional scaling (MDS) still showed a significant task difference in AIT, but not LIP, indicating that beyond increasing discriminability, attention also causes a nonlinear warping of representation space in AIT. Despite single-cell attentional modulations in both areas, our data show that attentional modulations of population representations are weaker in LIP, likely due to a need to maintain veridical representations for visuomotor control. PMID:29876521

  19. Vagal stimulation targets select populations of intrinsic cardiac neurons to control neurally induced atrial fibrillation.

    Science.gov (United States)

    Salavatian, Siamak; Beaumont, Eric; Longpré, Jean-Philippe; Armour, J Andrew; Vinet, Alain; Jacquemet, Vincent; Shivkumar, Kalyanam; Ardell, Jeffrey L

    2016-11-01

    Mediastinal nerve stimulation (MNS) reproducibly evokes atrial fibrillation (AF) by excessive and heterogeneous activation of intrinsic cardiac (IC) neurons. This study evaluated whether preemptive vagus nerve stimulation (VNS) impacts MNS-induced evoked changes in IC neural network activity to thereby alter susceptibility to AF. IC neuronal activity in the right atrial ganglionated plexus was directly recorded in anesthetized canines (n = 8) using a linear microelectrode array concomitant with right atrial electrical activity in response to: 1) epicardial touch or great vessel occlusion vs. 2) stellate or vagal stimulation. From these stressors, post hoc analysis (based on the Skellam distribution) defined IC neurons so recorded as afferent, efferent, or convergent (afferent and efferent inputs) local circuit neurons (LCN). The capacity of right-sided MNS to modify IC activity in the induction of AF was determined before and after preemptive right (RCV)- vs. left (LCV)-sided VNS (15 Hz, 500 μs; 1.2× bradycardia threshold). Neuronal (n = 89) activity at baseline (0.11 ± 0.29 Hz) increased during MNS-induced AF (0.51 ± 1.30 Hz; P neuronal synchrony increased during neurally induced AF, a local neural network response mitigated by preemptive VNS. These antiarrhythmic effects persisted post-VNS for, on average, 26 min. In conclusion, VNS preferentially targets convergent LCNs and their interactive coherence to mitigate the potential for neurally induced AF. The antiarrhythmic properties imposed by VNS exhibit memory. Copyright © 2016 the American Physiological Society.

  20. Vagal stimulation targets select populations of intrinsic cardiac neurons to control neurally induced atrial fibrillation

    Science.gov (United States)

    Salavatian, Siamak; Beaumont, Eric; Longpré, Jean-Philippe; Armour, J. Andrew; Vinet, Alain; Jacquemet, Vincent; Shivkumar, Kalyanam

    2016-01-01

    Mediastinal nerve stimulation (MNS) reproducibly evokes atrial fibrillation (AF) by excessive and heterogeneous activation of intrinsic cardiac (IC) neurons. This study evaluated whether preemptive vagus nerve stimulation (VNS) impacts MNS-induced evoked changes in IC neural network activity to thereby alter susceptibility to AF. IC neuronal activity in the right atrial ganglionated plexus was directly recorded in anesthetized canines (n = 8) using a linear microelectrode array concomitant with right atrial electrical activity in response to: 1) epicardial touch or great vessel occlusion vs. 2) stellate or vagal stimulation. From these stressors, post hoc analysis (based on the Skellam distribution) defined IC neurons so recorded as afferent, efferent, or convergent (afferent and efferent inputs) local circuit neurons (LCN). The capacity of right-sided MNS to modify IC activity in the induction of AF was determined before and after preemptive right (RCV)- vs. left (LCV)-sided VNS (15 Hz, 500 μs; 1.2× bradycardia threshold). Neuronal (n = 89) activity at baseline (0.11 ± 0.29 Hz) increased during MNS-induced AF (0.51 ± 1.30 Hz; P < 0.001). Convergent LCNs were preferentially activated by MNS. Preemptive RCV reduced MNS-induced changes in LCN activity (by 70%) while mitigating MNS-induced AF (by 75%). Preemptive LCV reduced LCN activity by 60% while mitigating AF potential by 40%. IC neuronal synchrony increased during neurally induced AF, a local neural network response mitigated by preemptive VNS. These antiarrhythmic effects persisted post-VNS for, on average, 26 min. In conclusion, VNS preferentially targets convergent LCNs and their interactive coherence to mitigate the potential for neurally induced AF. The antiarrhythmic properties imposed by VNS exhibit memory. PMID:27591222

  1. Circulating red cell-derived microparticles in human malaria.

    Science.gov (United States)

    Nantakomol, Duangdao; Dondorp, Arjen M; Krudsood, Srivicha; Udomsangpetch, Rachanee; Pattanapanyasat, Kovit; Combes, Valery; Grau, Georges E; White, Nicholas J; Viriyavejakul, Parnpen; Day, Nicholas P J; Chotivanich, Kesinee

    2011-03-01

    In patients with falciparum malaria, plasma concentrations of cell-derived microparticles correlate with disease severity. Using flow cytometry, we quantified red blood cell-derived microparticles (RMPs) in patients with malaria and identified the source and the factors associated with production. RMP concentrations were increased in patients with Plasmodium falciparum (n = 29; median, 457 RMPs/μL [range, 13-4,342 RMPs/μL]), Plasmodium vivax (n = 5; median, 409 RMPs/μL [range, 281-503/μL]), and Plasmodium malariae (n = 2; median, 163 RMPs/μL [range, 127-200 RMPs/μL]) compared with those in healthy subjects (n = 11; median, 8 RMPs/μL [range, 3-166 RMPs/μL]; P = .01). RMP concentrations were highest in patients with severe falciparum malaria (P = .01). Parasitized red cells produced >10 times more RMPs than did unparasitized cells, but the overall majority of RMPs still derived from uninfected red blood cells (URBCs). In cultures, RMP production increased as the parasites matured. Hemin and parasite products induced RMP production in URBCs, which was inhibited by N-acetylcysteine, suggesting heme-mediated oxidative stress as a pathway for the generation of RMPs.

  2. Up-regulation of neural indicators of empathic concern in an offender population.

    Science.gov (United States)

    Arbuckle, Nathan L; Shane, Matthew S

    2017-08-01

    Empathic concern has traditionally been conceived of as a spontaneous reaction to others experiencing pain or distress. As such, the potential role of more deliberate control over empathic responses has frequently been overlooked. The present fMRI study evaluated the role of such deliberate control in empathic concern by examining the extent to which a sample of offenders recruited through probation/parole could voluntarily modulate their neural activity to another person in pain. Offenders were asked to either passively view pictures of other people in painful or non-painful situations, or to actively modulate their level of concern for the person in pain. During passive viewing of painful versus non-painful pictures, offenders showed minimal neural activity in regions previously linked to empathy for pain (e.g., dorsal anterior cingulate cortex and bilateral insula). However, when instructed to try to increase their concern for the person in pain, offenders demonstrated significant increases within these regions. These findings are consistent with recent theories of empathy as motivational in nature, and suggest that limitations in empathic concern may include a motivational component.

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

    Directory of Open Access Journals (Sweden)

    Sebastian Werneburg

    2015-05-01

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

  4. Functional Properties of Human Stem Cell-Derived Neurons in Health and Disease

    Directory of Open Access Journals (Sweden)

    Jason P. Weick

    2016-01-01

    Full Text Available Stem cell-derived neurons from various source materials present unique model systems to examine the fundamental properties of central nervous system (CNS development as well as the molecular underpinnings of disease phenotypes. In order to more accurately assess potential therapies for neurological disorders, multiple strategies have been employed in recent years to produce neuronal populations that accurately represent in vivo regional and transmitter phenotypes. These include new technologies such as direct conversion of somatic cell types into neurons and glia which may accelerate maturation and retain genetic hallmarks of aging. In addition, novel forms of genetic manipulations have brought human stem cells nearly on par with those of rodent with respect to gene targeting. For neurons of the CNS, the ultimate phenotypic characterization lies with their ability to recapitulate functional properties such as passive and active membrane characteristics, synaptic activity, and plasticity. These features critically depend on the coordinated expression and localization of hundreds of ion channels and receptors, as well as scaffolding and signaling molecules. In this review I will highlight the current state of knowledge regarding functional properties of human stem cell-derived neurons, with a primary focus on pluripotent stem cells. While significant advances have been made, critical hurdles must be overcome in order for this technology to support progression toward clinical applications.

  5. Artificial Neural Network (ANN) Model to Predict Depression among Geriatric Population at a Slum in Kolkata, India.

    Science.gov (United States)

    Sau, Arkaprabha; Bhakta, Ishita

    2017-05-01

    Depression is one of the most important causes of mortality and morbidity among the geriatric population. Although, the aging brain is more vulnerable to depression, it cannot be considered as physiological and an inevitable part of ageing. Various sociodemographic and morbidity factors are responsible for the depression among them. Using Artificial Neural Network (ANN) model depression can be predicted from various sociodemographic variables and co morbid conditions even at community level by the grass root level health care workers. To predict depression among geriatric population from sociodemographic and morbidity attributes using ANN. An observational descriptive study with cross-sectional design was carried out at a slum under the service area of Bagbazar Urban Health and Training Centre (UHTC) in Kolkata. Among 126 elderlies under Bagbazar UHTC, 105 were interviewed using predesigned and pretested schedule. Depression status was assessed using 30 item Geriatric Depression Scale. WEKA 3.8.0 was used to develop the ANN model and test its performance. Prevalence of depression among the study population was 45.7%. Various sociodemographic variables like age, gender, literacy, living spouse, working status, personal income, family type, substance abuse and co morbid conditions like visual problem, mobility problem, hearing problem and sleeping problem were taken into consideration to develop the model. Prediction accuracy of this ANN model was 97.2%. Depression among geriatric population can be predicted accurately using ANN model from sociodemographic and morbidity attributes.

  6. Balancing Ethical Pros and Cons of Stem Cell Derived Gametes.

    Science.gov (United States)

    Segers, Seppe; Mertes, Heidi; de Wert, Guido; Dondorp, Wybo; Pennings, Guido

    2017-07-01

    In this review we aim to provide an overview of the most important ethical pros and cons of stem cell derived gametes (SCD-gametes), as a contribution to the debate about reproductive tissue engineering. Derivation of gametes from stem cells holds promising applications both for research and for clinical use in assisted reproduction. We explore the ethical issues connected to gametes derived from embryonic stem cells (both patient specific and non-patient specific) as well as those related to gametes derived from induced pluripotent stem cells. The technology of SCD-gametes raises moral concerns of how reproductive autonomy relates to issues of embryo destruction, safety, access, and applications beyond clinical infertility.

  7. Stem-cell-derived products: an FDA update.

    Science.gov (United States)

    Moos, Malcolm

    2008-12-01

    The therapeutic potential of products derived from stem cells of various types has prompted increasing research and development and public attention. Initiation of human clinical trials in the not-too-distant future is now a realistic possibility. It is, therefore, important to weigh the potential benefits against known, theoretical and totally unsuspected risks in light of current knowledge to ensure that subjects participating in these trials are afforded the most reasonable balance possible between potential risks and potential benefits. There are no apparent differences in fundamental, qualitative biological characteristics between stem-cell-derived products and other cellular therapies regulated by the United States Food and Drug Administration (FDA). Existing authorities can, therefore, be applied. Nevertheless, these products do have properties that require careful evaluation.

  8. Delivery of chemotherapeutic drugs in tumour cell-derived microparticles.

    Science.gov (United States)

    Tang, Ke; Zhang, Yi; Zhang, Huafeng; Xu, Pingwei; Liu, Jing; Ma, Jingwei; Lv, Meng; Li, Dapeng; Katirai, Foad; Shen, Guan-Xin; Zhang, Guimei; Feng, Zuo-Hua; Ye, Duyun; Huang, Bo

    2012-01-01

    Cellular microparticles are vesicular plasma membrane fragments with a diameter of 100-1,000 nanometres that are shed by cells in response to various physiological and artificial stimuli. Here we demonstrate that tumour cell-derived microparticles can be used as vectors to deliver chemotherapeutic drugs. We show that tumour cells incubated with chemotherapeutic drugs package these drugs into microparticles, which can be collected and used to effectively kill tumour cells in murine tumour models without typical side effects. We describe several mechanisms involved in this process, including uptake of drug-containing microparticles by tumour cells, synthesis of additional drug-packaging microparticles by these cells that contribute to the cytotoxic effect and the inhibition of drug efflux from tumour cells. This study highlights a novel drug delivery strategy with potential clinical application.

  9. Cell-derived microparticles in haemostasis and vascular medicine.

    Science.gov (United States)

    Burnier, Laurent; Fontana, Pierre; Kwak, Brenda R; Angelillo-Scherrer, Anne

    2009-03-01

    Considerable interest for cell-derived microparticles has emerged, pointing out their essential role in haemostatic response and their potential as disease markers, but also their implication in a wide range of physiological and pathological processes. They derive from different cell types including platelets - the main source of microparticles - but also from red blood cells, leukocytes and endothelial cells, and they circulate in blood. Despite difficulties encountered in analyzing them and disparities of results obtained with a wide range of methods, microparticle generation processes are now better understood. However, a generally admitted definition of microparticles is currently lacking. For all these reasons we decided to review the literature regarding microparticles in their widest definition, including ectosomes and exosomes, and to focus mainly on their role in haemostasis and vascular medicine.

  10. Trophoblast lineage cells derived from human induced pluripotent stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Ying, E-mail: ying.chen@hc.msu.edu [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, 333 Bostwick NE, Grand Rapids, MI 49503 (United States); Wang, Kai; Chandramouli, Gadisetti V.R. [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, 333 Bostwick NE, Grand Rapids, MI 49503 (United States); Knott, Jason G. [Developmental Epigenetics Laboratory, Department of Animal Science, Michigan State University (United States); Leach, Richard, E-mail: Richard.leach@hc.msu.edu [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, 333 Bostwick NE, Grand Rapids, MI 49503 (United States); Department of Obstetrics, Gynecology and Women’s Health, Spectrum Health Medical Group (United States)

    2013-07-12

    Highlights: •Epithelial-like phenotype of trophoblast lineage cells derived from human iPS cells. •Trophoblast lineage cells derived from human iPS cells exhibit trophoblast function. •Trophoblasts from iPS cells provides a proof-of-concept in regenerative medicine. -- Abstract: Background: During implantation, the blastocyst trophectoderm attaches to the endometrial epithelium and continues to differentiate into all trophoblast subtypes, which are the major components of a placenta. Aberrant trophoblast proliferation and differentiation are associated with placental diseases. However, due to ethical and practical issues, there is almost no available cell or tissue source to study the molecular mechanism of human trophoblast differentiation, which further becomes a barrier to the study of the pathogenesis of trophoblast-associated diseases of pregnancy. In this study, our goal was to generate a proof-of-concept model for deriving trophoblast lineage cells from induced pluripotency stem (iPS) cells from human fibroblasts. In future studies the generation of trophoblast lineage cells from iPS cells established from patient’s placenta will be extremely useful for studying the pathogenesis of individual trophoblast-associated diseases and for drug testing. Methods and results: Combining iPS cell technology with BMP4 induction, we derived trophoblast lineage cells from human iPS cells. The gene expression profile of these trophoblast lineage cells was distinct from fibroblasts and iPS cells. These cells expressed markers of human trophoblasts. Furthermore, when these cells were differentiated they exhibited invasive capacity and placental hormone secretive capacity, suggesting extravillous trophoblasts and syncytiotrophoblasts. Conclusion: Trophoblast lineage cells can be successfully derived from human iPS cells, which provide a proof-of-concept tool to recapitulate pathogenesis of patient placental trophoblasts in vitro.

  11. Trophoblast lineage cells derived from human induced pluripotent stem cells

    International Nuclear Information System (INIS)

    Chen, Ying; Wang, Kai; Chandramouli, Gadisetti V.R.; Knott, Jason G.; Leach, Richard

    2013-01-01

    Highlights: •Epithelial-like phenotype of trophoblast lineage cells derived from human iPS cells. •Trophoblast lineage cells derived from human iPS cells exhibit trophoblast function. •Trophoblasts from iPS cells provides a proof-of-concept in regenerative medicine. -- Abstract: Background: During implantation, the blastocyst trophectoderm attaches to the endometrial epithelium and continues to differentiate into all trophoblast subtypes, which are the major components of a placenta. Aberrant trophoblast proliferation and differentiation are associated with placental diseases. However, due to ethical and practical issues, there is almost no available cell or tissue source to study the molecular mechanism of human trophoblast differentiation, which further becomes a barrier to the study of the pathogenesis of trophoblast-associated diseases of pregnancy. In this study, our goal was to generate a proof-of-concept model for deriving trophoblast lineage cells from induced pluripotency stem (iPS) cells from human fibroblasts. In future studies the generation of trophoblast lineage cells from iPS cells established from patient’s placenta will be extremely useful for studying the pathogenesis of individual trophoblast-associated diseases and for drug testing. Methods and results: Combining iPS cell technology with BMP4 induction, we derived trophoblast lineage cells from human iPS cells. The gene expression profile of these trophoblast lineage cells was distinct from fibroblasts and iPS cells. These cells expressed markers of human trophoblasts. Furthermore, when these cells were differentiated they exhibited invasive capacity and placental hormone secretive capacity, suggesting extravillous trophoblasts and syncytiotrophoblasts. Conclusion: Trophoblast lineage cells can be successfully derived from human iPS cells, which provide a proof-of-concept tool to recapitulate pathogenesis of patient placental trophoblasts in vitro

  12. Cell-derived microparticles promote coagulation after moderate exercise.

    Science.gov (United States)

    Sossdorf, Maik; Otto, Gordon P; Claus, Ralf A; Gabriel, Holger H W; Lösche, Wolfgang

    2011-07-01

    Cell-derived procoagulant microparticles (MP) might be able to contribute to exercise-induced changes in blood hemostasis. This study aimed to examine (i) the concentration and procoagulant activity of cell-derived MP after a moderate endurance exercise and (ii) the differences in the release, clearance, and activity of MP before and after exercise between trained and untrained individuals. All subjects performed a single bout of physical exercise on a bicycle ergometer for 90 min at 80% of their individual anaerobic threshold. MP were identified and quantified by flow cytometry measurements. Procoagulant activity of MP was measured by a prothrombinase activity assay as well as tissue factor-induced fibrin formation in MP-containing plasma. At baseline, no differences were observed for the absolute number and procoagulant activities of MP between trained and untrained subjects. However, trained individuals had a lower number of tissue factor-positive monocyte-derived MP compared with untrained individuals. In trained subjects, exercise induced a significant increase in the number of MP derived from platelets, monocytes, and endothelial cells, with maximum values at 45 min after exercise and returned to basal levels at 2 h after exercise. Untrained subjects revealed a similar increase in platelet-derived MP, but their level was still increased at 2 h after exercise, indicating a reduced clearance compared with trained individuals. Procoagulant activities of MP were increased immediately after exercise and remained elevated up to 2 h after exercise. We conclude that increased levels of MP were found in healthy individuals after an acute bout of exercise, that the amount of circulating MP contributes to an exercise-induced increase of hemostatic potential, and that there were differences in kinetic and dynamic characteristics between trained and untrained individuals.

  13. A constructive mean-field analysis of multi-population neural networks with random synaptic weights and stochastic inputs.

    Science.gov (United States)

    Faugeras, Olivier; Touboul, Jonathan; Cessac, Bruno

    2009-01-01

    We deal with the problem of bridging the gap between two scales in neuronal modeling. At the first (microscopic) scale, neurons are considered individually and their behavior described by stochastic differential equations that govern the time variations of their membrane potentials. They are coupled by synaptic connections acting on their resulting activity, a nonlinear function of their membrane potential. At the second (mesoscopic) scale, interacting populations of neurons are described individually by similar equations. The equations describing the dynamical and the stationary mean-field behaviors are considered as functional equations on a set of stochastic processes. Using this new point of view allows us to prove that these equations are well-posed on any finite time interval and to provide a constructive method for effectively computing their unique solution. This method is proved to converge to the unique solution and we characterize its complexity and convergence rate. We also provide partial results for the stationary problem on infinite time intervals. These results shed some new light on such neural mass models as the one of Jansen and Rit (1995): their dynamics appears as a coarse approximation of the much richer dynamics that emerges from our analysis. Our numerical experiments confirm that the framework we propose and the numerical methods we derive from it provide a new and powerful tool for the exploration of neural behaviors at different scales.

  14. Differentiation and functional incorporation of embryonic stem cell-derived GABAergic interneurons in the dentate gyrus of mice with temporal lobe epilepsy.

    Science.gov (United States)

    Maisano, Xu; Litvina, Elizabeth; Tagliatela, Stephanie; Aaron, Gloster B; Grabel, Laura B; Naegele, Janice R

    2012-01-04

    Cell therapies for neurological disorders require an extensive knowledge of disease-associated neuropathology and procedures for generating neurons for transplantation. In many patients with severe acquired temporal lobe epilepsy (TLE), the dentate gyrus exhibits sclerosis and GABAergic interneuron degeneration. Mounting evidence suggests that therapeutic benefits can be obtained by transplanting fetal GABAergic progenitors into the dentate gyrus in rodents with TLE, but the scarcity of human fetal cells limits applicability in patient populations. In contrast, virtually limitless quantities of neural progenitors can be obtained from embryonic stem (ES) cells. ES cell-based therapies for neurological repair in TLE require evidence that the transplanted neurons integrate functionally and replace cell types that degenerate. To address these issues, we transplanted mouse ES cell-derived neural progenitors (ESNPs) with ventral forebrain identities into the hilus of the dentate gyrus of mice with TLE and evaluated graft differentiation, mossy fiber sprouting, cellular morphology, and electrophysiological properties of the transplanted neurons. In addition, we compared electrophysiological properties of the transplanted neurons with endogenous hilar interneurons in mice without TLE. The majority of transplanted ESNPs differentiated into GABAergic interneuron subtypes expressing calcium-binding proteins parvalbumin, calbindin, or calretinin. Global suppression of mossy fiber sprouting was not observed; however, ESNP-derived neurons formed dense axonal arborizations in the inner molecular layer and throughout the hilus. Whole-cell hippocampal slice electrophysiological recordings and morphological analyses of the transplanted neurons identified five basic types; most with strong after-hyperpolarizations and smooth or sparsely spiny dendritic morphologies resembling endogenous hippocampal interneurons. Moreover, intracellular recordings of spontaneous EPSCs indicated that

  15. Regenerative medicine for Parkinson's disease using differentiated nerve cells derived from human buccal fat pad stem cells.

    Science.gov (United States)

    Takahashi, Haruka; Ishikawa, Hiroshi; Tanaka, Akira

    2017-04-01

    The purpose of this study was to evaluate the utility of human adipose stem cells derived from the buccal fat pad (hBFP-ASCs) for nerve regeneration. Parkinson's disease (PD) is a neurodegenerative disorder characterized by progressive death of dopaminergic neurons. PD is a candidate disease for cell replacement therapy because it has no fundamental therapeutic methods. We examined the properties of neural-related cells induced from hBFP-ASCs as a cell source for PD treatment. hBFP-ASCs were cultured in neurogenic differentiation medium for about 2 weeks. After the morphology of hBFP-ASCs changed to neural-like cells, the medium was replaced with neural maintenance medium. Cells differentiated from hBFP-ASCs showed neuron-like structures and expressed neuron markers (β3-tubulin, neurofilament 200, and microtubule-associated protein 2), an astrocyte marker (glial fibrillary acidic protein), or dopaminergic neuron-related marker (tyrosine hydroxylase). Induced neural cells were transplanted into a 6-hydroxydopamine (6-OHDA)-lesioned rat hemi-parkinsonian model. At 4 weeks after transplantation, 6-OHDA-lesioned rats were subjected to apomorphine-induced rotation analysis. The transplanted cells survived in the brain of rats as dopaminergic neural cells. No tumor formation was found after cell transplantation. We demonstrated differentiation of hBFP-ASCs into neural cells, and that transplantation of these neural cells improved the symptoms of model rats. Our results suggest that neurons differentiated from hBFP-ASCs would be applicable to cell replacement therapy of PD.

  16. Novel Mutation of LRP6 Identified in Chinese Han Population Links Canonical WNT Signaling to Neural Tube Defects.

    Science.gov (United States)

    Shi, Zhiwen; Yang, Xueyan; Li, Bin-Bin; Chen, Shuxia; Yang, Luming; Cheng, Liangping; Zhang, Ting; Wang, Hongyan; Zheng, Yufang

    2018-01-15

    Neural tube defects (NTDs), the second most frequent cause of human congenital abnormalities, are debilitating birth defects due to failure of neural tube closure. It has been shown that noncanonical WNT/planar cell polarity (PCP) signaling is required for convergent extension (CE), the initiation step of neural tube closure (NTC). But the effect of canonical WNT//β-catenin signaling during NTC is still elusive. LRP6 (low density lipoprotein receptor related proteins 6) was identified as a co-receptor for WNT/β-catenin signaling, but recent studies showed that it also can mediate WNT/PCP signaling. In this study, we screened mutations in the LRP6 gene in 343 NTDs and 215 ethnically matched normal controls of Chinese Han population. Three rare missense mutations (c.1514A>G, p.Y505C); c.2984A>G, p.D995G; and c.4280C>A, p.P1427Q) of the LRP6 gene were identified in Chinese NTD patients. The Y505C mutation is a loss-of-function mutation on both WNT/β-catenin and PCP signaling. The D995G mutation only partially lost inhibition on PCP signaling without affecting WNT/β-catenin signaling. The P1427Q mutation dramatically increased WNT/β-catenin signaling but only mildly loss of inhibition on PCP signaling. All three mutations failed to rescue CE defects caused by lrp6 morpholino oligos knockdown in zebrafish. Of interest, when overexpressed, D995G did not induce any defects, but Y505C and P1427Q caused more severe CE defects in zebrafish. Our results suggested that over-active canonical WNT signaling induced by gain-of-function mutation in LRP6 could also contribute to human NTDs, and a balanced WNT/β-catenin and PCP signaling is probably required for proper neural tube development. Birth Defects Research 110:63-71, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  17. Coding of time-dependent stimuli in homogeneous and heterogeneous neural populations.

    Science.gov (United States)

    Beiran, Manuel; Kruscha, Alexandra; Benda, Jan; Lindner, Benjamin

    2018-04-01

    We compare the information transmission of a time-dependent signal by two types of uncoupled neuron populations that differ in their sources of variability: i) a homogeneous population whose units receive independent noise and ii) a deterministic heterogeneous population, where each unit exhibits a different baseline firing rate ('disorder'). Our criterion for making both sources of variability quantitatively comparable is that the interspike-interval distributions are identical for both systems. Numerical simulations using leaky integrate-and-fire neurons unveil that a non-zero amount of both noise or disorder maximizes the encoding efficiency of the homogeneous and heterogeneous system, respectively, as a particular case of suprathreshold stochastic resonance. Our findings thus illustrate that heterogeneity can render similarly profitable effects for neuronal populations as dynamic noise. The optimal noise/disorder depends on the system size and the properties of the stimulus such as its intensity or cutoff frequency. We find that weak stimuli are better encoded by a noiseless heterogeneous population, whereas for strong stimuli a homogeneous population outperforms an equivalent heterogeneous system up to a moderate noise level. Furthermore, we derive analytical expressions of the coherence function for the cases of very strong noise and of vanishing intrinsic noise or heterogeneity, which predict the existence of an optimal noise intensity. Our results show that, depending on the type of signal, noise as well as heterogeneity can enhance the encoding performance of neuronal populations.

  18. A prospective crossover comparison of neurally adjusted ventilatory assist and pressure-support ventilation in a pediatric and neonatal intensive care unit population.

    LENUS (Irish Health Repository)

    Breatnach, Cormac

    2012-02-01

    OBJECTIVE: To compare neurally adjusted ventilatory assist ventilation with pressure-support ventilation. DESIGN: Prospective, crossover comparison study. SETTING: Tertiary care pediatric and neonatal intensive care unit. PATIENTS: Sixteen ventilated infants and children: mean age = 9.7 months (range = 2 days-4 yrs) and mean weight = 6.2 kg (range = 2.4-13.7kg). INTERVENTIONS: A modified nasogastric tube was inserted and correct positioning was confirmed. Patients were ventilated in pressure-support mode with a pneumatic trigger for a 30-min period and then in neurally adjusted ventilatory assist mode for up to 4 hrs. MEASUREMENTS AND MAIN RESULTS: Data collected for comparison included activating trigger (neural vs. pneumatic), peak and mean airway pressures, expired minute and tidal volumes, heart rate, respiratory rate, pulse oximetry, end-tidal CO2 and arterial blood gases. Synchrony was improved in neurally adjusted ventilatory assist mode with 65% (+\\/-21%) of breaths triggered neurally vs. 35% pneumatically (p < .001) and 85% (+\\/-8%) of breaths cycled-off neurally vs. 15% pneumatically (p = .0001). The peak airway pressure in neurally adjusted ventilatory assist mode was significantly lower than in pressure-support mode with a 28% decrease in pressure after 30 mins (p = .003) and 32% decrease after 3 hrs (p < .001). Mean airway pressure was reduced by 11% at 30 mins (p = .13) and 9% at 3 hrs (p = .31) in neurally adjusted ventilatory assist mode although this did not reach statistical significance. Patient hemodynamics and gas exchange remained stable for the study period. No adverse patient events or device effects were noted. CONCLUSIONS: In a neonatal and pediatric intensive care unit population, ventilation in neurally adjusted ventilatory assist mode was associated with improved patient-ventilator synchrony and lower peak airway pressure when compared with pressure-support ventilation with a pneumatic trigger. Ventilating patients in this new mode

  19. A stem cell medium containing neural stimulating factor induces a pancreatic cancer stem-like cell-enriched population

    Science.gov (United States)

    WATANABE, YUSAKU; YOSHIMURA, KIYOSHI; YOSHIKAWA, KOICHI; TSUNEDOMI, RYOICHI; SHINDO, YOSHITARO; MATSUKUMA, SOU; MAEDA, NORIKO; KANEKIYO, SHINSUKE; SUZUKI, NOBUAKI; KURAMASU, ATSUO; SONODA, KOUHEI; TAMADA, KOJI; KOBAYASHI, SEI; SAYA, HIDEYUKI; HAZAMA, SHOICHI; OKA, MASAAKI

    2014-01-01

    Cancer stem cells (CSCs) have been studied for their self-renewal capacity and pluripotency, as well as their resistance to anticancer therapy and their ability to metastasize to distant organs. CSCs are difficult to study because their population is quite low in tumor specimens. To overcome this problem, we established a culture method to induce a pancreatic cancer stem-like cell (P-CSLC)-enriched population from human pancreatic cancer cell lines. Human pancreatic cancer cell lines established at our department were cultured in CSC-inducing media containing epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), neural cell survivor factor-1 (NSF-1), and N-acetylcysteine. Sphere cells were obtained and then transferred to a laminin-coated dish and cultured for approximately two months. The surface markers, gene expression, aldehyde dehydrogenase (ALDH) activity, cell cycle, and tumorigenicity of these induced cells were examined for their stem cell-like characteristics. The population of these induced cells expanded within a few months. The ratio of CD24high, CD44high, epithelial specific antigen (ESA) high, and CD44variant (CD44v) high cells in the induced cells was greatly enriched. The induced cells stayed in the G0/G1 phase and demonstrated mesenchymal and stemness properties. The induced cells had high tumorigenic potential. Thus, we established a culture method to induce a P-CSLCenriched population from human pancreatic cancer cell lines. The CSLC population was enriched approximately 100-fold with this method. Our culture method may contribute to the precise analysis of CSCs and thus support the establishment of CSC-targeting therapy. PMID:25118635

  20. Pluripotent stem cell-derived neural stem cells: From basic research to applications

    OpenAIRE

    Otsu, Masahiro; Nakayama, Takashi; Inoue, Nobuo

    2014-01-01

    Basic research on pluripotent stem cells is designed to enhance understanding of embryogenesis, whereas applied research is designed to develop novel therapies and prevent diseases. Attainment of these goals has been enhanced by the establishment of embryonic stem cell lines, the technological development of genomic reprogramming to generate induced-pluripotent stem cells, and improvements in vitro techniques to manipulate stem cells. This review summarizes the techniques required to generate...

  1. Plasticity of Calcium Signaling Cascades in Human Embryonic Stem Cell-Derived Neural Precursors

    Czech Academy of Sciences Publication Activity Database

    Forostyak, Oksana; Romanyuk, Nataliya; Verkhratsky, A.; Syková, Eva; Dayanithi, Govindan

    2013-01-01

    Roč. 22, č. 10 (2013), s. 1506-1521 ISSN 1547-3287 R&D Projects: GA ČR GAP304/11/2373; GA ČR(CZ) GBP304/12/G069 Grant - others:FP7(XE) PITN-GA-2008-214003 project AXREGEN; FP7(XE) PITN-GA-2009-237956 project EdU-GLIA Institutional support: RVO:68378041 Keywords : human embryonic stem cells * voltage-operated Ca2+ channels * spontaneous Ca2+ oscillations Subject RIV: FH - Neurology Impact factor: 4.202, year: 2013

  2. Physiology of CA2+ signaling in human embryonic stem cell-derived neural precursors

    Czech Academy of Sciences Publication Activity Database

    Forostyak, Oksana; Romanyuk, Nataliya; Syková, Eva; Dayanithi, Govindan

    2011-01-01

    Roč. 59, S1 (2011), S119-S119 ISSN 0894-1491. [European meeting on Glia l Cells in Health and Disease /10./. 13.09.2011-17.09.2011, Prague] R&D Projects: GA ČR(CZ) GAP303/11/0192 Institutional research plan: CEZ:AV0Z50390703 Keywords : calcium signaling * ATP * calcium channels Subject RIV: FH - Neurology

  3. Neural markers of emotional face perception across psychotic disorders and general population.

    Science.gov (United States)

    Sabharwal, Amri; Kotov, Roman; Szekely, Akos; Leung, Hoi-Chung; Barch, Deanna M; Mohanty, Aprajita

    2017-07-01

    There is considerable variation in negative and positive symptoms of psychosis, global functioning, and emotional face perception (EFP), not only in schizophrenia but also in other psychotic disorders and healthy individuals. However, EFP impairment and its association with worse symptoms and global functioning have been examined largely in the domain of schizophrenia. The present study adopted a dimensional approach to examine the association of behavioral and neural measures of EFP with symptoms of psychosis and global functioning across individuals with schizophrenia spectrum (SZ; N = 28) and other psychotic (OP; N = 29) disorders, and never-psychotic participants (NP; N = 21). Behavioral and functional MRI data were recorded as participants matched emotional expressions of faces and geometrical shapes. Lower accuracy and increased activity in early visual regions, hippocampus, and amygdala during emotion versus shape matching were associated with higher negative, but not positive, symptoms and lower global functioning, across all participants. This association remained even after controlling for group-related (SZ, OP, and NP) variance, dysphoria, and antipsychotic medication status, except in amygdala. Furthermore, negative symptoms mediated the relationship between behavioral and brain EFP measures and global functioning. This study provides some of the first evidence supporting the specific relationship of EFP measures with negative symptoms and global functioning across psychotic and never-psychotic samples, and transdiagnostically across different psychotic disorders. Present findings help bridge the gap between basic EFP-related neuroscience research and clinical research in psychosis, and highlight EFP as a potential symptom-specific marker that tracks global functioning. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

  4. Immune surveillance properties of human NK cell-derived exosomes.

    Science.gov (United States)

    Lugini, Luana; Cecchetti, Serena; Huber, Veronica; Luciani, Francesca; Macchia, Gianfranco; Spadaro, Francesca; Paris, Luisa; Abalsamo, Laura; Colone, Marisa; Molinari, Agnese; Podo, Franca; Rivoltini, Licia; Ramoni, Carlo; Fais, Stefano

    2012-09-15

    Exosomes are nanovesicles released by normal and tumor cells, which are detectable in cell culture supernatant and human biological fluids, such as plasma. Functions of exosomes released by "normal" cells are not well understood. In fact, several studies have been carried out on exosomes derived from hematopoietic cells, but very little is known about NK cell exosomes, despite the importance of these cells in innate and adaptive immunity. In this paper, we report that resting and activated NK cells, freshly isolated from blood of healthy donors, release exosomes expressing typical protein markers of NK cells and containing killer proteins (i.e., Fas ligand and perforin molecules). These nanovesicles display cytotoxic activity against several tumor cell lines and activated, but not resting, immune cells. We also show that NK-derived exosomes undergo uptake by tumor target cells but not by resting PBMC. Exosomes purified from plasma of healthy donors express NK cell markers, including CD56+ and perforin, and exert cytotoxic activity against different human tumor target cells and activated immune cells as well. The results of this study propose an important role of NK cell-derived exosomes in immune surveillance and homeostasis. Moreover, this study supports the use of exosomes as an almost perfect example of biomimetic nanovesicles possibly useful in future therapeutic approaches against various diseases, including tumors.

  5. Cells derived from young bone marrow alleviate renal aging.

    Science.gov (United States)

    Yang, Hai-Chun; Rossini, Michele; Ma, Li-Jun; Zuo, Yiqin; Ma, Ji; Fogo, Agnes B

    2011-11-01

    Bone marrow-derived stem cells may modulate renal injury, but the effects may depend on the age of the stem cells. Here we investigated whether bone marrow from young mice attenuates renal aging in old mice. We radiated female 12-mo-old 129SvJ mice and reconstituted them with bone marrow cells (BMC) from either 8-wk-old (young-to-old) or 12-mo-old (old-to-old) male mice. Transfer of young BMC resulted in markedly decreased deposition of collagen IV in the mesangium and less β-galactosidase staining, an indicator of cell senescence. These changes paralleled reduced expression of plasminogen activator inhibitor-1 (PAI-1), PDGF-B (PDGF-B), the transdifferentiation marker fibroblast-specific protein-1 (FSP-1), and senescence-associated p16 and p21. Tubulointerstitial and glomerular cells derived from the transplanted BMC did not show β-galactosidase activity, but after 6 mo, there were more FSP-1-expressing bone marrow-derived cells in old-to-old mice compared with young-to-old mice. Young-to-old mice also exhibited higher expression of the anti-aging gene Klotho and less phosphorylation of IGF-1 receptor β. Taken together, these data suggest that young bone marrow-derived cells can alleviate renal aging in old mice. Direct parenchymal reconstitution by stem cells, paracrine effects from adjacent cells, and circulating anti-aging molecules may mediate the aging of the kidney.

  6. Large-scale generation of cell-derived nanovesicles

    Science.gov (United States)

    Jo, W.; Kim, J.; Yoon, J.; Jeong, D.; Cho, S.; Jeong, H.; Yoon, Y. J.; Kim, S. C.; Gho, Y. S.; Park, J.

    2014-09-01

    Exosomes are enclosed compartments that are released from cells and that can transport biological contents for the purpose of intercellular communications. Research into exosomes is hindered by their rarity. In this article, we introduce a device that uses centrifugal force and a filter with micro-sized pores to generate a large quantity of cell-derived nanovesicles. The device has a simple polycarbonate structure to hold the filter, and operates in a common centrifuge. Nanovesicles are similar in size and membrane structure to exosomes. Nanovesicles contain intracellular RNAs ranging from microRNA to mRNA, intracellular proteins, and plasma membrane proteins. The quantity of nanovesicles produced using the device is 250 times the quantity of naturally secreted exosomes. Also, the quantity of intracellular contents in nanovesicles is twice that in exosomes. Nanovesicles generated from murine embryonic stem cells can transfer RNAs to target cells. Therefore, this novel device and the nanovesicles that it generates are expected to be used in exosome-related research, and can be applied in various applications such as drug delivery and cell-based therapy.

  7. [Cell-derived microparticles unveil their fibrinolytic and proteolytic function].

    Science.gov (United States)

    Doeuvre, Loïc; Angles-Cano, Eduardo

    2009-01-01

    Cell-derived microparticles (MP) are membrane microvesicles, 0.1-1 microm in size, shed by cells following activation or during apoptosis in a variety of pathological conditions. MPs released by blood cells or by vascular endothelial cells display molecular signatures that allow their identification and functional characterization. In addition, they provide tissue factor (TF) and a procoagulant phospholipid surface. Therefore, at present, the most strongly established applied research on MPs is their procoagulant activity as a determinant of thrombotic risk in various clinical conditions. Previous studies have indicated that MPs derived from malignant cells express matrix metalloproteinases, urokinase and its receptor (uPA/uPAR) that, in the presence of plasminogen, may act in concert to degrade extracellular matrix proteins. Recently, it was shown that MPs from TNFa-stimulated endothelial cells served as a surface for interaction with plasminogen and its conversion into plasmin by the uPA/uPAR system expressed at their surface. This capacity of MPs to promote plasmin generation confers them a new profibrinolytic and proteolytic function that may be of relevance in fibrinolysis, cell migration, angiogenesis, dissemination of malignant cells, cell detachment and apoptosis.

  8. Application of convolutional artificial neural networks to echocardiograms for differentiating congenital heart diseases in a pediatric population

    Science.gov (United States)

    Perrin, Douglas P.; Bueno, Alejandra; Rodriguez, Andrea; Marx, Gerald R.; del Nido, Pedro J.

    2017-03-01

    In this paper we describe a pilot study, where machine learning methods are used to differentiate between congenital heart diseases. Our approach was to apply convolutional neural networks (CNNs) to echocardiographic images from five different pediatric populations: normal, coarctation of the aorta (CoA), hypoplastic left heart syndrome (HLHS), transposition of the great arteries (TGA), and single ventricle (SV). We used a single network topology that was trained in a pairwise fashion in order to evaluate the potential to differentiate between patient populations. In total we used 59,151 echo frames drawn from 1,666 clinical sequences. Approximately 80% of the data was used for training, and the remainder for validation. Data was split at sequence boundaries to avoid having related images in the training and validation sets. While training was done with echo images/frames, evaluation was performed for both single frame discrimination as well as sequence discrimination (by majority voting). In total 10 networks were generated and evaluated. Unlike other domains where this network topology has been used, in ultrasound there is low visual variation between classes. This work shows the potential for CNNs to be applied to this low-variation domain of medical imaging for disease discrimination.

  9. Comparison of tumour age response to radiation for cells derived from tissue culture or solid tumours

    International Nuclear Information System (INIS)

    Keng, P.C.; Siemann, D.W.; Rochester Univ., NY; Rochester Univ., NY; Wheeler, K.T.

    1984-01-01

    Direct comparison of the cell age response of 9L and KHT tumour cells derived either from tissue culture or solid tumours was achieved. Cells from dissociated KHT and 9L tumours (the latter implanted either subcutaneously or intracerebrally) and cells from tissue culture were separated into homogenous sized populations by centrifugal elutriation. In both tumour models these homogeneous sized populations correspond to populations enriched at different stages of the cell cycle. The survival of these elutriated cell populations was measured after a single dose of Cs-137 gamma rays. For cells isolated from 9L solid tumours, there was little variation in radiosensitivity throughout the cell cycle; however, a very small but significant increase in resistance was found in late G 1 cells. This lack of a large variation in radiosensitivity through the cell cycle for 9L cells from solid tumours also was seen in 9L cells growing in monolayer tissue culture. When similar experiments were performed using the KHT sarcoma tumour model, the results showed that KHT cells in vitro exhibited a fairly conventional increase in radioresistance in both mid G 1 and late S. However, the cell age response of KHT cells from solid tumours was different; particularly in the late S and G 2 + M phases. (author)

  10. Characterization and modulation of canine mast cell derived eicosanoids

    Science.gov (United States)

    Lin, Tzu-Yin; London, Cheryl A.

    2013-01-01

    Mast cells play an important role in both innate and acquired immunity as well as several pathological conditions including allergy, arthritis and neoplasia. They influence these processes by producing a variety of mediators including cytokines, chemokines and eicosanoids. Very little is currently known about the spectrum of inflammatory mediators, particularly eicosanoids (prostaglandins and leukotrienes), produced by canine mast cells. This is important since modulating mast cell derived eicosanoids may help in the treatment of autoimmune and inflammatory disorders. The purpose of this study was to investigate the spectrum of eicosanoids produced by normal canine mast cells and to evaluate the effects of cytokines and non-steroidal anti-inflammatory mediators (NSAIDS) on eicosanoid production and release. Canine bone marrow derived cultured mast cells (cBMCMCs) expressed COX-1, COX-2, and 5-LOX and synthesized and released PGD2, PGE2, LTB4, and LTC4 following activation by a variety of stimuli. The selective COX-2 NSAIDs carprofen (Rimadyl®) and deracoxib (Deramaxx®) inhibited PGD2 and PGE2 production but only slightly inhibited LTB4 and LTC4. The mixed COX-1/COX-2 inhibitor piroxicam blocked PGD2 and PGE2 production, but upregulated LTC4 following treatment while tepoxilan (Zubrin®), a pan COX/LOX inhibitor, markedly reduced the production of all eicosanoids. The LOX inhibitor nordihydroguaiaretic acid (NDGA) prevented LTB4/LTC4 release and BMBMC degranulation. Pre-incubation of cBMCMCs with IL-4 and SCF sensitized these cells to degranulation in response to substance P. In conclusion, canine BMCMCs produce an array of eicosanoids similar to those produced by mast cells from other species. Tepoxilan appeared to be the most effective NSAID for blocking eicosanoid production and thus may be useful for modulating mast cell mediated responses in dogs. PMID:20036014

  11. Influence of red blood cell-derived microparticles upon vasoregulation.

    Science.gov (United States)

    Said, Ahmed S; Doctor, Allan

    2017-10-01

    Here we review recent data and the evolving understanding of the role of red blood cell-derived microparticles (RMPs) in normal physiology and in disease progression. Microparticles (MPs) are small membrane vesicles derived from various parent cell types. MPs are produced in response to a variety of stimuli through several cytoskeletal and membrane phospholipid changes. MPs have been investigated as potential biomarkers for multiple disease processes and are thought to have biological effects, most notably in: promotion of coagulation, production and handling of reactive oxygen species, immune modulation, angiogenesis, and in apoptosis. Specifically, RMPs are produced normally during RBC maturation and their production is accelerated during processing and storage for transfusion. Several factors during RBC storage are known to trigger RMP production, including: increased intracellular calcium, increased potassium leakage, and energy failure with ATP depletion. Of note, RMP composition differs from that of intact RBCs, and the nature and composition of RMP components are affected by both storage duration and the character of storage solutions. Recognised RMP bioactivities include: promotion of coagulation, immune modulation, and promotion of endothelial adhesion, as well as influence upon vasoregulation via nitric oxide (NO) scavenging. Of particular relevance, RMPs are more avid NO scavengers than intact RBCs and this feature has been proposed as a mechanism for the impaired oxygen delivery homeostasis that has been observed following transfusion. Preliminary human studies demonstrate that circulating RMP abundance increases with RBC transfusion and is associated with altered plasma vasoactivity and abnormal vasoregulation. In summary, RMPs are submicron particles released from stored RBCs, with demonstrated vasoactive properties that appear to disturb oxygen delivery homeostasis. The clinical impact of RMPs in transfusion recipients is an area of continued

  12. Red cell-derived microparticles (RMP) as haemostatic agent.

    Science.gov (United States)

    Jy, Wenche; Johansen, Max E; Bidot, Carlos; Horstman, Lawrence L; Ahn, Yeon S

    2013-10-01

    Among circulating cell-derived microparticles, those derived from red cells (RMP) have been least well investigated. To exploit potential haemostatic benefit of RMP, we developed a method of producing them in quantity, and here report on their haemostatic properties. High-pressure extrusion of washed RBC was employed to generate RMP. RMP were identified and enumerated by flow cytometry. Their size distribution was assessed by Doppler electrophoretic light scattering analysis (DELSA). Interaction with platelets was studied by platelet aggregometry, and shear-dependent adhesion by Diamed IMPACT-R. Thrombin generation and tissue factor (TF) expression was also measured. The effect of RMP on blood samples of patients with bleeding disorders was investigated ex vivo by thromboelastography (TEG). Haemostatic efficacy in vivo was assessed by measuring reduction of blood loss and bleeding time in rats and rabbits. RMP have mean diameter of 0.45 µm and 50% of them exhibit annexin V binding, a proxy for procoagulant phospholipids (PL). No TF could be detected by flow cytometry. At saturating concentrations of MPs, RMP generated thrombin robustly but after longer delay compared to PMP and EMP. RMP enhanced platelet adhesion and aggregation induced by low-dose ADP or AA. In TEG study, RMP corrected or improved haemostatic defects in blood of patients with platelet and coagulation disorders. RMP reduced bleeding time and blood loss in thrombocytopenic rabbits (busulfan-treated) and in Plavix-treated rats. In conclusion, RMP has broad haemostatic activity, enhancing both primary (platelet) and secondary (coagulation) haemostasis, suggesting potential use as haemostatic agent for treatment of bleeding.

  13. L-type calcium channels refine the neural population code of sound level

    Science.gov (United States)

    Grimsley, Calum Alex; Green, David Brian

    2016-01-01

    The coding of sound level by ensembles of neurons improves the accuracy with which listeners identify how loud a sound is. In the auditory system, the rate at which neurons fire in response to changes in sound level is shaped by local networks. Voltage-gated conductances alter local output by regulating neuronal firing, but their role in modulating responses to sound level is unclear. We tested the effects of L-type calcium channels (CaL: CaV1.1–1.4) on sound-level coding in the central nucleus of the inferior colliculus (ICC) in the auditory midbrain. We characterized the contribution of CaL to the total calcium current in brain slices and then examined its effects on rate-level functions (RLFs) in vivo using single-unit recordings in awake mice. CaL is a high-threshold current and comprises ∼50% of the total calcium current in ICC neurons. In vivo, CaL activates at sound levels that evoke high firing rates. In RLFs that increase monotonically with sound level, CaL boosts spike rates at high sound levels and increases the maximum firing rate achieved. In different populations of RLFs that change nonmonotonically with sound level, CaL either suppresses or enhances firing at sound levels that evoke maximum firing. CaL multiplies the gain of monotonic RLFs with dynamic range and divides the gain of nonmonotonic RLFs with the width of the RLF. These results suggest that a single broad class of calcium channels activates enhancing and suppressing local circuits to regulate the sensitivity of neuronal populations to sound level. PMID:27605536

  14. Functional Differences in Engineered Myocardium from Embryonic Stem Cell-Derived versus Neonatal Cardiomyocytes

    NARCIS (Netherlands)

    Feinberg, Adam W.; Ripplinger, Crystal M.; van der Meer, Peter; Sheehy, Sean P.; Domian, Ibrahim; Chien, Kenneth R.; Parker, Kevin Kit

    2013-01-01

    Stem cell-derived cardiomyocytes represent unique tools for cell-and tissue-based regenerative therapies, drug discovery and safety, and studies of fundamental heart-failure mechanisms. However, the degree to which stem cell-derived cardiomyocytes compare to mature cardiomyocytes is often debated.

  15. Predicting symptomatic cerebral vasospasm after aneurysmal subarachnoid hemorrhage with an artificial neural network in a pediatric population.

    Science.gov (United States)

    Skoch, Jesse; Tahir, Rizwan; Abruzzo, Todd; Taylor, John M; Zuccarello, Mario; Vadivelu, Sudhakar

    2017-12-01

    Artificial neural networks (ANN) are increasingly applied to complex medical problem solving algorithms because their outcome prediction performance is superior to existing multiple regression models. ANN can successfully identify symptomatic cerebral vasospasm (SCV) in adults presenting after aneurysmal subarachnoid hemorrhage (aSAH). Although SCV is unusual in children with aSAH, the clinical consequences are severe. Consequently, reliable tools to predict patients at greatest risk for SCV may have significant value. We applied ANN modeling to a consecutive cohort of pediatric aSAH cases to assess its ability to predict SCV. A retrospective chart review was conducted to identify patients met study inclusion criteria. The median age for aSAH patients was 15 years. Ten underwent surgical clipping and 6 underwent endovascular coiling for definitive treatment. One patient experienced SCV and 15 did not. The ANN applied here was able to accurately predict all 16 outcomes. The mean strength of prediction for those who did not exhibit SCV was - 0.86. The strength for the one patient who did exhibit SCV was 0.93. Adult-derived aneurysmal SAH value nodes can be applied to a simple AAN model to accurately predict SCV in children presenting with aSAH. Further work is needed to determine if ANN models can prospectively predict SCV in the pediatric aSAH population in toto; adapted to include mycotic, traumatic, and flow-related origins as well.

  16. Association of main folate metabolic pathway gene polymorphisms with neural tube defects in Han population of Northern China.

    Science.gov (United States)

    Fang, Yulian; Zhang, Ruiping; Zhi, Xiufang; Zhao, Linsheng; Cao, Lirong; Wang, Yizheng; Cai, Chunquan

    2018-04-01

    Neural tube defects (NTDs) are one of the most prevalent and the most severe congenital malformations worldwide. Studies have confirmed that folic acid supplementation could effectively reduce NTDs risk, but the genetic mechanism remains unclear. In this study, we explored association of single nucleotide polymorphisms (SNP) within folate metabolic pathway genes with NTDs in Han population of Northern China. We performed a case-control study to compare genotype and allele distributions of SNPs in 152 patients with NTDs and 169 controls. A total of 16 SNPs within five genes were genotyped by the Sequenom MassARRAY assay. Our results indicated that three SNPs associated significantly with NTDs (P<0.05). For rs2236225 within MTHFD1, children with allele A or genotype AA had a high NTDs risk (OR=1.500, 95%CI=1.061~2.120; OR=2.862, 95%CI=1.022~8.015, respectively). For rs1801133 within MTHFR, NTDs risk markedly increased in patients with allele T or genotype TT (OR=1.552, 95%CI=1.130~2.131; OR=2.344, 95%CI=1.233~4.457, respectively). For rs1801394 within MTRR, children carrying allele G and genotype GG had a higher NTDs risk (OR=1.533, 95%CI=1.102~2.188; OR=2.355, 95%CI=1.044~5.312, respectively). Our results suggest that rs2236225 of MTHFD1 gene, rs1801133 of MTHFR gene and rs1801394 of MTRR gene were associated with NTDs in Han population of Northern China.

  17. Differentiation and molecular profiling of human embryonic stem cell-derived corneal epithelial cells.

    Science.gov (United States)

    Brzeszczynska, J; Samuel, K; Greenhough, S; Ramaesh, K; Dhillon, B; Hay, D C; Ross, J A

    2014-06-01

    It has been suggested that the isolation of scalable populations of limbal stem cells may lead to radical changes in ocular therapy. In particular, the derivation and transplantation of corneal stem cells from these populations may result in therapies providing clinical normality of the diseased or damaged cornea. Although feasible in theory, the lack of donor material in sufficient quantity and quality currently limits such a strategy. A potential scalable source of corneal cells could be derived from pluripotent stem cells (PSCs). We developed an in vitro and serum-free corneal differentiation model which displays significant promise. Our stepwise differentiation model was designed with reference to development and gave rise to cells which displayed similarities to epithelial progenitor cells which can be specified to cells displaying a corneal epithelial phenotype. We believe our approach is novel, provides a robust model of human development and in the future, may facilitate the generation of corneal epithelial cells that are suitable for clinical use. Additionally, we demonstrate that following continued cell culture, stem cell-derived corneal epithelial cells undergo transdifferentiation and exhibit squamous metaplasia and therefore, also offer an in vitro model of disease.

  18. The emergence of two anti-phase oscillatory neural populations in a computational model of the Parkinsonian globus pallidus

    Directory of Open Access Journals (Sweden)

    Robert John Merrison-Hort

    2013-12-01

    Full Text Available Experiments in rodent models of Parkinson's Disease have demonstrated a prominent increase of oscillatory firing patterns in neurons within the Parkinsonian globus pallidus (GP which may underlie some of the motor symptoms of the disease. There are two main pathways from the cortex to GP: via the striatum and via the subthalamic nucleus (STN, but it is not known how these inputs sculpt the pathological pallidal firing patterns. To study this we developed a novel neural network model of conductance-based spiking pallidal neurons with cortex-modulated input from STN neurons. Our results support the hypothesis that entrainment occurs primarily via the subthalamic pathway. We find that as a result of the interplay between excitatory input from the STN and mutual inhibitory coupling between GP neurons, a homogeneous population of GP neurons demonstrates a self-organising dynamical behaviour where two groups of neurons emerge: one spiking in-phase with the cortical rhythm and the other in anti-phase. This finding mirrors what is seen in recordings from the GP of rodents that have had Parkinsonism induced via brain lesions. Our model also includes downregulation of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN channels in response to burst firing of GP neurons, since this has been suggested as a possible mechanism for the emergence of Parkinsonian activity. We found that the downregulation of HCN channels provides even better correspondence with experimental data but that it is not essential in order for the two groups of oscillatory neurons to appear. We discuss how the influence of inhibitory striatal input will strengthen our results.

  19. Tcf3 represses Wnt-β-catenin signaling and maintains neural stem cell population during neocortical development.

    Directory of Open Access Journals (Sweden)

    Atsushi Kuwahara

    Full Text Available During mouse neocortical development, the Wnt-β-catenin signaling pathway plays essential roles in various phenomena including neuronal differentiation and proliferation of neural precursor cells (NPCs. Production of the appropriate number of neurons without depletion of the NPC population requires precise regulation of the balance between differentiation and maintenance of NPCs. However, the mechanism that suppresses Wnt signaling to prevent premature neuronal differentiation of NPCs is poorly understood. We now show that the HMG box transcription factor Tcf3 (also known as Tcf7l1 contributes to this mechanism. Tcf3 is highly expressed in undifferentiated NPCs in the mouse neocortex, and its expression is reduced in intermediate neuronal progenitors (INPs committed to the neuronal fate. We found Tcf3 to be a repressor of Wnt signaling in neocortical NPCs in a reporter gene assay. Tcf3 bound to the promoter of the proneural bHLH gene Neurogenin1 (Neurog1 and repressed its expression. Consistent with this, Tcf3 repressed neuronal differentiation and increased the self-renewal activity of NPCs. We also found that Wnt signal stimulation reduces the level of Tcf3, and increases those of Tcf1 (also known as Tcf7 and Lef1, positive mediators of Wnt signaling, in NPCs. Together, these results suggest that Tcf3 antagonizes Wnt signaling in NPCs, thereby maintaining their undifferentiated state in the neocortex and that Wnt signaling promotes the transition from Tcf3-mediated repression to Tcf1/Lef1-mediated enhancement of Wnt signaling, constituting a positive feedback loop that facilitates neuronal differentiation.

  20. Developmental changes in electrophysiological characteristics of human induced Pluripotent Stem Cell-derived cardiomyocytes

    Science.gov (United States)

    Ben-Ari, Meital; Naor, Shulamit; Zeevi-Levin, Naama; Schick, Revital; Ben Jehuda, Ronen; Reiter, Irina; Raveh, Amit; Grijnevitch, Inna; Barak, Omri; Rosen, Michael R.; Weissman, Amir; Binah, Ofer

    2016-01-01

    Background Previous studies proposed that throughout differentiation of human induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CMs) only 3 types of action potentials (AP) exist: nodal, atrial and ventricular-like. Objective To investigate whether there are precisely 3 phenotypes or a continuum exists among them, we tested 2 hypotheses: (1) during culture development a cardiac precursor cell is present that - depending on age - can evolve into the 3 phenotypes. (2) The predominant pattern is early prevalence of nodal phenotype, transient appearance of atrial phenotype, evolution to ventricular phenotype, and persistence of transitional phenotypes. Methods To test these hypotheses we: (1) performed FACS analysis of nodal, atrial and ventricular markers; (2) recorded AP from 280 7-to-95 day old iPSC-CMs; (3) analyzed AP characteristics. Results The major findings were: (1) FACS analysis of 30 and 60-day old cultures showed that an iPSC-CMs population shifts from nodal into atrial/ventricular phenotype, while including significant transitional populations.(2) The AP population did not consist of 3 distinct phenotypes; (3) Culture aging was associated with a shift from nodal to ventricular dominance, with a transient (57–70 days) appearance of atrial phenotype; (4) Beat Rate Variability was more prominent in nodal than ventricular cardiomyocytes while If density increased in older cultures. Conclusions From the onset of development the iPSC-CMs population includes nodal, atrial and ventricular AP and a broad spectrum of transitional phenotypes. The most readily distinguishable phenotype is atrial which appears only transiently, yet dominates at 57–70 days of evolution. PMID:27639456

  1. Developmental changes in electrophysiological characteristics of human-induced pluripotent stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Ben-Ari, Meital; Naor, Shulamit; Zeevi-Levin, Naama; Schick, Revital; Ben Jehuda, Ronen; Reiter, Irina; Raveh, Amit; Grijnevitch, Inna; Barak, Omri; Rosen, Michael R; Weissman, Amir; Binah, Ofer

    2016-12-01

    Previous studies proposed that throughout differentiation of human induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CMs), only 3 types of action potentials (APs) exist: nodal-, atrial-, and ventricular-like. To investigate whether there are precisely 3 phenotypes or a continuum exists among them, we tested 2 hypotheses: (1) During culture development a cardiac precursor cell is present that-depending on age-can evolve into the 3 phenotypes. (2) The predominant pattern is early prevalence of a nodal phenotype, transient appearance of an atrial phenotype, evolution to a ventricular phenotype, and persistence of transitional phenotypes. To test these hypotheses, we (1) performed fluorescence-activated cell sorting analysis of nodal, atrial, and ventricular markers; (2) recorded APs from 280 7- to 95-day-old iPSC-CMs; and (3) analyzed AP characteristics. The major findings were as follows: (1) fluorescence-activated cell sorting analysis of 30- and 60-day-old cultures showed that an iPSC-CMs population shifts from the nodal to the atrial/ventricular phenotype while including significant transitional populations; (2) the AP population did not consist of 3 phenotypes; (3) culture aging was associated with a shift from nodal to ventricular dominance, with a transient (57-70 days) appearance of the atrial phenotype; and (4) beat rate variability was more prominent in nodal than in ventricular cardiomyocytes, while pacemaker current density increased in older cultures. From the onset of development in culture, the iPSC-CMs population includes nodal, atrial, and ventricular APs and a broad spectrum of transitional phenotypes. The most readily distinguishable phenotype is atrial, which appears only transiently yet dominates at 57-70 days of evolution. Copyright © 2016 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

  2. Potential Therapies by Stem Cell-Derived Exosomes in CNS Diseases: Focusing on the Neurogenic Niche

    Directory of Open Access Journals (Sweden)

    Alejandro Luarte

    2016-01-01

    Full Text Available Neurodegenerative disorders are one of the leading causes of death and disability and one of the biggest burdens on health care systems. Novel approaches using various types of stem cells have been proposed to treat common neurodegenerative disorders such as Alzheimer’s Disease, Parkinson’s Disease, or stroke. Moreover, as the secretome of these cells appears to be of greater benefit compared to the cells themselves, the extracellular components responsible for its therapeutic benefit have been explored. Stem cells, as well as most cells, release extracellular vesicles such as exosomes, which are nanovesicles able to target specific cell types and thus to modify their function by delivering proteins, lipids, and nucleic acids. Exosomes have recently been tested in vivo and in vitro as therapeutic conveyors for the treatment of diseases. As such, they could be engineered to target specific populations of cells within the CNS. Considering the fact that many degenerative brain diseases have an impact on adult neurogenesis, we discuss how the modulation of the adult neurogenic niches may be a therapeutic target of stem cell-derived exosomes. These novel approaches should be examined in cellular and animal models to provide better, more effective, and specific therapeutic tools in the future.

  3. Preclinical Studies of Induced Pluripotent Stem Cell-Derived Astrocyte Transplantation in ALS

    Science.gov (United States)

    2012-10-01

    Pluripotent Stem Cell -Derived Astrocyte Transplantation in ALS PRINCIPAL INVESTIGATOR: Nicholas J. Maragakis, M.D...Pluripotent Stem Cell -Derived Astrocyte Transplantation in ALS 5b. GRANT NUMBER W81XWH-10-1-0520 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d...into astrocytes following transplantation. 15. SUBJECT TERMS Stem Cells , iPS cells, astrocytes, familial ALS 16. SECURITY CLASSIFICATION OF

  4. Stem Cell-Derived Exosome in Cardiovascular Diseases: Macro Roles of Micro Particles.

    Science.gov (United States)

    Yuan, Ye; Du, Weijie; Liu, Jiaqi; Ma, Wenya; Zhang, Lai; Du, Zhimin; Cai, Benzhi

    2018-01-01

    The stem cell-based therapy has emerged as the promising therapeutic strategies for cardiovascular diseases (CVDs). Recently, increasing evidence suggest stem cell-derived active exosomes are important communicators among cells in the heart via delivering specific substances to the adjacent/distant target cells. These exosomes and their contents such as certain proteins, miRNAs and lncRNAs exhibit huge beneficial effects on preventing heart damage and promoting cardiac repair. More importantly, stem cell-derived exosomes are more effective and safer than stem cell transplantation. Therefore, administration of stem cell-derived exosomes will expectantly be an alternative stem cell-based therapy for the treatment of CVDs. Furthermore, modification of stem cell-derived exosomes or artificial synthesis of exosomes will be the new therapeutic tools for CVDs in the future. In addition, stem cell-derived exosomes also have been implicated in the diagnosis and prognosis of CVDs. In this review, we summarize the current advances of stem cell-derived exosome-based treatment and prognosis for CVDs, including their potential benefits, underlying mechanisms and limitations, which will provide novel insights of exosomes as a new tool in clinical therapeutic translation in the future.

  5. Potential roles of cell-derived microparticles in ischemic brain disease.

    Science.gov (United States)

    Horstman, Lawrence L; Jy, Wenche; Bidot, Carlos J; Nordberg, Mary L; Minagar, Alireza; Alexander, J Steven; Kelley, Roger E; Ahn, Yeon S

    2009-10-01

    The objective of this study is to review the role of cell-derived microparticles in ischemic cerebrovascular diseases. An extensive PubMed search of literature pertaining to this study was performed in April 2009 using specific keyword search terms related to cell-derived microparticles and ischemic stroke. Some references are not cited here as it is not possible to be all inclusive or due to space limitation. Cell-derived microparticles are small membranous vesicles released from the plasma membranes of platelets, leukocytes, red cells and endothelial cells in response to diverse biochemical agents or mechanical stresses. They are the main carriers of circulating tissue factor, the principal initiator of intravascular thrombosis, and are implicated in a variety of thrombotic and inflammatory disorders. This review outlines evidence suggesting that cell-derived microparticles are involved predominantly with microvascular, as opposed to macrovascular, thrombosis. More specifically, cell-derived microparticles may substantially contribute to ischemic brain disease in several settings, as well as to neuroinflammatory conditions. If further work confirms this hypothesis, novel therapeutic strategies for minimizing cell-derived microparticles-mediated ischemia are available or can be developed, as discussed.

  6. Β-amyloid 1-42 oligomers impair function of human embryonic stem cell-derived forebrain cholinergic neurons.

    Directory of Open Access Journals (Sweden)

    Linn Wicklund

    Full Text Available Cognitive impairment in Alzheimer's disease (AD patients is associated with a decline in the levels of growth factors, impairment of axonal transport and marked degeneration of basal forebrain cholinergic neurons (BFCNs. Neurogenesis persists in the adult human brain, and the stimulation of regenerative processes in the CNS is an attractive prospect for neuroreplacement therapy in neurodegenerative diseases such as AD. Currently, it is still not clear how the pathophysiological environment in the AD brain affects stem cell biology. Previous studies investigating the effects of the β-amyloid (Aβ peptide on neurogenesis have been inconclusive, since both neurogenic and neurotoxic effects on progenitor cell populations have been reported. In this study, we treated pluripotent human embryonic stem (hES cells with nerve growth factor (NGF as well as with fibrillar and oligomeric Aβ1-40 and Aβ1-42 (nM-µM concentrations and thereafter studied the differentiation in vitro during 28-35 days. The process applied real time quantitative PCR, immunocytochemistry as well as functional studies of intracellular calcium signaling. Treatment with NGF promoted the differentiation into functionally mature BFCNs. In comparison to untreated cells, oligomeric Aβ1-40 increased the number of functional neurons, whereas oligomeric Aβ1-42 suppressed the number of functional neurons. Interestingly, oligomeric Aβ exposure did not influence the number of hES cell-derived neurons compared with untreated cells, while in contrast fibrillar Aβ1-40 and Aβ1-42 induced gliogenesis. These findings indicate that Aβ1-42 oligomers may impair the function of stem cell-derived neurons. We propose that it may be possible for future AD therapies to promote the maturation of functional stem cell-derived neurons by altering the brain microenvironment with trophic support and by targeting different aggregation forms of Aβ.

  7. Epigenetic profiles in children with a neural tube defect; a case-control study in two populations

    NARCIS (Netherlands)

    L. Stolk (Lisette); M.I. Both (Marieke); N.H. van Mill (Nina); M.M.P.J. Verbiest (Michael); P.H.C. Eilers (Paul); H. Zhu (Huiping); L. Suarez (Lucina); A.G. Uitterlinden (André); R.P.M. Steegers-Theunissen (Régine)

    2013-01-01

    textabstractFolate deficiency is implicated in the causation of neural tube defects (NTDs). The preventive effect of periconceptional folic acid supplement use is partially explained by the treatment of a deranged folate-dependent one carbon metabolism, which provides methyl groups for

  8. The chemokine receptor CCR1 is identified in mast cell-derived exosomes.

    Science.gov (United States)

    Liang, Yuting; Qiao, Longwei; Peng, Xia; Cui, Zelin; Yin, Yue; Liao, Huanjin; Jiang, Min; Li, Li

    2018-01-01

    Mast cells are important effector cells of the immune system, and mast cell-derived exosomes carrying RNAs play a role in immune regulation. However, the molecular function of mast cell-derived exosomes is currently unknown, and here, we identify differentially expressed genes (DEGs) in mast cells and exosomes. We isolated mast cells derived exosomes through differential centrifugation and screened the DEGs from mast cell-derived exosomes, using the GSE25330 array dataset downloaded from the Gene Expression Omnibus database. Biochemical pathways were analyzed by Gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway on the online tool DAVID. DEGs-associated protein-protein interaction networks (PPIs) were constructed using the STRING database and Cytoscape software. The genes identified from these bioinformatics analyses were verified by qRT-PCR and Western blot in mast cells and exosomes. We identified 2121 DEGs (843 up and 1278 down-regulated genes) in HMC-1 cell-derived exosomes and HMC-1 cells. The up-regulated DEGs were classified into two significant modules. The chemokine receptor CCR1 was screened as a hub gene and enriched in cytokine-mediated signaling pathway in module one. Seven genes, including CCR1, CD9, KIT, TGFBR1, TLR9, TPSAB1 and TPSB2 were screened and validated through qRT-PCR analysis. We have achieved a comprehensive view of the pivotal genes and pathways in mast cells and exosomes and identified CCR1 as a hub gene in mast cell-derived exosomes. Our results provide novel clues with respect to the biological processes through which mast cell-derived exosomes modulate immune responses.

  9. Foetal stem cell derivation & characterization for osteogenic lineage

    Directory of Open Access Journals (Sweden)

    A Mangala Gowri

    2013-01-01

    Full Text Available Background & objectives: Mesencymal stem cells (MSCs derived from foetal tissues present a multipotent progenitor cell source for application in tissue engineering and regenerative medicine. The present study was carried out to derive foetal mesenchymal stem cells from ovine source and analyze their differentiation to osteogenic linage to serve as an animal model to predict human applications. Methods: Isolation and culture of sheep foetal bone marrow cells were done and uniform clonally derived MSC population was collected. The cells were characterized using cytochemical, immunophenotyping, biochemical and molecular analyses. The cells with defined characteristics were differentiated into osteogenic lineages and analysis for differentiated cell types was done. The cells were analyzed for cell surface marker expression and the gene expression in undifferentiated and differentiated osteoblast was checked by reverse transcriptase PCR (RT PCR analysis and confirmed by sequencing using genetic analyzer. Results: Ovine foetal samples were processed to obtain mononuclear (MNC cells which on culture showed spindle morphology, a characteristic oval body with the flattened ends. MSC population CD45 - /CD14 - was cultured by limiting dilution to arrive at uniform spindle morphology cells and colony forming units. The cells were shown to be positive for surface markers such as CD44, CD54, integrinβ1, and intracellular collagen type I/III and fibronectin. The osteogenically induced MSCs were analyzed for alkaline phosphatase (ALP activity and mineral deposition. The undifferentiated MSCs expressed RAB3B, candidate marker for stemness in MSCs. The osteogenically induced and uninduced MSCs expressed collagen type I and MMP13 gene in osteogenic induced cells. Interpretation & conclusions: The protocol for isolation of ovine foetal bone marrow derived MSCs was simple to perform, and the cultural method of obtaining pure spindle morphology cells was established

  10. Involvement of multiple myeloma cell-derived exosomes in osteoclast differentiation

    Science.gov (United States)

    Raimondi, Lavinia; De Luca, Angela; Amodio, Nicola; Manno, Mauro; Raccosta, Samuele; Taverna, Simona; Bellavia, Daniele; Naselli, Flores; Fontana, Simona; Schillaci, Odessa; Giardino, Roberto; Fini, Milena; Tassone, Pierfrancesco; Santoro, Alessandra; De Leo, Giacomo; Giavaresi, Gianluca; Alessandro, Riccardo

    2015-01-01

    Bone disease is the most frequent complication in multiple myeloma (MM) resulting in osteolytic lesions, bone pain, hypercalcemia and renal failure. In MM bone disease the perfect balance between bone-resorbing osteoclasts (OCs) and bone-forming osteoblasts (OBs) activity is lost in favour of OCs, thus resulting in skeletal disorders. Since exosomes have been described for their functional role in cancer progression, we here investigate whether MM cell-derived exosomes may be involved in OCs differentiation. We show that MM cells produce exosomes which are actively internalized by Raw264.7 cell line, a cellular model of osteoclast formation. MM cell-derived exosomes positively modulate pre-osteoclast migration, through the increasing of CXCR4 expression and trigger a survival pathway. MM cell-derived exosomes play a significant pro-differentiative role in murine Raw264.7 cells and human primary osteoclasts, inducing the expression of osteoclast markers such as Cathepsin K (CTSK), Matrix Metalloproteinases 9 (MMP9) and Tartrate-resistant Acid Phosphatase (TRAP). Pre-osteoclast treated with MM cell-derived exosomes differentiate in multinuclear OCs able to excavate authentic resorption lacunae. Similar results were obtained with exosomes derived from MM patient's sera. Our data indicate that MM-exosomes modulate OCs function and differentiation. Further studies are needed to identify the OCs activating factors transported by MM cell-derived exosomes. PMID:25944696

  11. Predicting carotid artery disease and plaque instability from cell-derived microparticles.

    Science.gov (United States)

    Wekesa, A L; Cross, K S; O'Donovan, O; Dowdall, J F; O'Brien, O; Doyle, M; Byrne, L; Phelan, J P; Ross, M D; Landers, R; Harrison, M

    2014-11-01

    Cell-derived microparticles (MPs) are small plasma membrane-derived vesicles shed from circulating blood cells and may act as novel biomarkers of vascular disease. We investigated the potential of circulating MPs to predict (a) carotid plaque instability and (b) the presence of advanced carotid disease. This pilot study recruited carotid disease patients (aged 69.3 ± 1.2 years [mean ± SD], 69% male, 90% symptomatic) undergoing endarterectomy (n = 42) and age- and sex-matched controls (n = 73). Plaques were classified as stable (n = 25) or unstable (n = 16) post surgery using immunohistochemistry. Blood samples were analysed for MP subsets and molecular biomarkers. Odds ratios (OR) are expressed per standard deviation biomarker increase. Endothelial MP (EMP) subsets, but not any vascular, inflammatory, or proteolytic molecular biomarker, were higher (p < .05) in the unstable than the stable plaque patients. The area under the receiver operator characteristic curve for CD31(+)41(-) EMP in discriminating an unstable plaque was 0.73 (0.56-0.90, p < .05). CD31(+)41(-) EMP predicted plaque instability (OR = 2.19, 1.08-4.46, p < .05) and remained significant in a multivariable model that included transient ischaemic attack symptom status. Annexin V(+) MP, platelet MP (PMP) subsets, and C-reactive protein were higher (p < .05) in cases than controls. Annexin V(+) MP (OR = 3.15, 1.49-6.68), soluble vascular cell adhesion molecule-1 (OR = 1.64, 1.03-2.59), and previous smoking history (OR = 3.82, 1.38-10.60) independently (p < .05) predicted the presence of carotid disease in a multivariable model. EMP may have utility in predicting plaque instability in carotid patients and annexin V(+) MPs may predict the presence of advanced carotid disease in aging populations, independent of established biomarkers. Copyright © 2014 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.

  12. Cardiotoxicity evaluation using human embryonic stem cells and induced pluripotent stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Zhao, Qi; Wang, Xijie; Wang, Shuyan; Song, Zheng; Wang, Jiaxian; Ma, Jing

    2017-03-09

    Cardiotoxicity remains an important concern in drug discovery. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have become an attractive platform to evaluate cardiotoxicity. However, the consistency between human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in prediction of cardiotoxicity has yet to be elucidated. Here we screened the toxicities of four representative drugs (E-4031, isoprenaline, quinidine, and haloperidol) using both hESC-CMs and hiPSC-CMs, combined with an impedance-based bioanalytical method. It showed that both hESC-CMs and hiPSC-CMs can recapitulate cardiotoxicity and identify the effects of well-characterized compounds. The combined platform of hPSC-CMs and an impedance-based bioanalytical method could improve preclinical cardiotoxicity screening, holding great potential for increasing drug development accuracy.

  13. T cell-derived Lymphotoxin is Essential for anti-HSV-1 Humoral Immune Response.

    Science.gov (United States)

    Yang, Kaiting; Liang, Yong; Sun, Zhichen; Xue, Diyuan; Xu, Hairong; Zhu, Mingzhao; Fu, Yang-Xin; Peng, Hua

    2018-05-09

    B cell-derived lymphotoxin (LT) is required for the development of follicular dendritic cell clusters for the formation of primary and secondary lymphoid follicles, but the role of T cell-derived LT in antibody response has not been well demonstrated. We observed that lymphotoxin-β-receptor (LTβR) signaling is essential for optimal humoral immune response and protection against an acute HSV-1 infection. Blocking the LTβR pathway caused poor maintenance of germinal center B (GC-B) cells and follicular helper T (Tfh) cells. Using bone marrow chimeric mice and adoptive transplantation, we determined that T cell-derived LT played an indispensable role in the humoral immune response to HSV-1. Up-regulation of IFNγ by the LTβR-Ig blockade impairs the sustainability of Tfh-like cells, thus leading to an impaired humoral immune response. Our findings have identified a novel role of T cell-derived LT in the humoral immune response against HSV-1 infection. IMPORTANCE Immunocompromised people are susceptible for HSV-1 infection and lethal recurrence, which could be inhibited by anti-HSV-1 humoral immune response in the host. This study sought to explore the role of T cell-derived LT in the anti-HSV-1 humoral immune response using LT-LTβR signaling deficient mice and the LTβR-Ig blockade. The data indicate that the T cell-derived LT may play an essential role in sustaining Tfh-like cells and ensure Tfh-like cells' migration into primary or secondary follicles for further maturation. This study provides insights for vaccine development against infectious diseases. Copyright © 2018 American Society for Microbiology.

  14. Solving the puzzle of pluripotent stem cell-derived cardiomyocyte maturation: piece by piece.

    Science.gov (United States)

    Lundy, David J; Lee, Desy S; Hsieh, Patrick C H

    2017-03-01

    There is a growing need for in vitro models which can serve as platforms for drug screening and basic research. Human adult cardiomyocytes cannot be readily obtained or cultured, and so pluripotent stem cell-derived cardiomyocytes appear to be an attractive option. Unfortunately, these cells are structurally and functionally immature-more comparable to foetal cardiomyocytes than adult. A recent study by Ruan et al ., provides new insights into accelerating the maturation process and takes us a step closer to solving the puzzle of pluripotent stem cell-derived cardiomyocyte maturation.

  15. Classification of follicular cell-derived thyroid cancer by global RNA profiling

    DEFF Research Database (Denmark)

    Rossing, Maria

    2013-01-01

    The incidence of thyroid cancer is increasing worldwide and thyroid nodules are a frequent clinical finding. Diagnosing follicular cell-derived cancers is, however, challenging both histopathologically and especially cytopathologically. The advent of high-throughput molecular technologies has...... profiling of follicular cell-derived thyroid cancers....... prompted many researchers to explore the transcriptome and, in recent years, also the miRNome in order to generate new molecular classifiers capable of classifying thyroid tumours more accurately than by conventional cytopathological and histopathological methods. This has led to a number of molecular...

  16. Epigenetic profiles in children with a neural tube defect; a case-control study in two populations.

    Directory of Open Access Journals (Sweden)

    Lisette Stolk

    Full Text Available Folate deficiency is implicated in the causation of neural tube defects (NTDs. The preventive effect of periconceptional folic acid supplement use is partially explained by the treatment of a deranged folate-dependent one carbon metabolism, which provides methyl groups for DNA-methylation as an epigenetic mechanism. Here, we hypothesize that variations in DNA-methylation of genes implicated in the development of NTDs and embryonic growth are part of the underlying mechanism. In 48 children with a neural tube defect and 62 controls from a Dutch case-control study and 34 children with a neural tube defect and 78 controls from a Texan case-control study, we measured the DNA-methylation levels of imprinted candidate genes (IGF2-DMR, H19, KCNQ1OT1 and non-imprinted genes (the LEKR/CCNL gene region associated with birth weight, and MTHFR and VANGL1 associated with NTD. We used the MassARRAY EpiTYPER assay from Sequenom for the assessment of DNA-methylation. Linear mixed model analysis was used to estimate associations between DNA-methylation levels of the genes and a neural tube defect. In the Dutch study group, but not in the Texan study group we found a significant association between the risk of having an NTD and DNA methylation levels of MTHFR (absolute decrease in methylation of -0.33% in cases, P-value = 0.001, and LEKR/CCNL (absolute increase in methylation: 1.36% in cases, P-value = 0.048, and a borderline significant association for VANGL (absolute increase in methylation: 0.17% in cases, P-value = 0.063. Only the association between MTHFR and NTD-risk remained significant after multiple testing correction. The associations in the Dutch study were not replicated in the Texan study. We conclude that the associations between NTDs and the methylation of the MTHFR gene, and maybe VANGL and LEKKR/CNNL, are in line with previous studies showing polymorphisms in the same genes in association with NTDs and embryonic development

  17. A novel mechanism of bacterial toxin transfer within host blood cell-derived microvesicles.

    Directory of Open Access Journals (Sweden)

    Anne-lie Ståhl

    2015-02-01

    Full Text Available Shiga toxin (Stx is the main virulence factor of enterohemorrhagic Escherichia coli, which are non-invasive strains that can lead to hemolytic uremic syndrome (HUS, associated with renal failure and death. Although bacteremia does not occur, bacterial virulence factors gain access to the circulation and are thereafter presumed to cause target organ damage. Stx was previously shown to circulate bound to blood cells but the mechanism by which it would potentially transfer to target organ cells has not been elucidated. Here we show that blood cell-derived microvesicles, shed during HUS, contain Stx and are found within patient renal cortical cells. The finding was reproduced in mice infected with Stx-producing Escherichia coli exhibiting Stx-containing blood cell-derived microvesicles in the circulation that reached the kidney where they were transferred into glomerular and peritubular capillary endothelial cells and further through their basement membranes followed by podocytes and tubular epithelial cells, respectively. In vitro studies demonstrated that blood cell-derived microvesicles containing Stx undergo endocytosis in glomerular endothelial cells leading to cell death secondary to inhibited protein synthesis. This study demonstrates a novel virulence mechanism whereby bacterial toxin is transferred within host blood cell-derived microvesicles in which it may evade the host immune system.

  18. Single-Cell Functional Analysis of Stem-Cell Derived Cardiomyocytes on Micropatterned Flexible Substrates

    NARCIS (Netherlands)

    Kijlstra, Jan David; Hu, Dongjian; van der Meer, Peter; Domian, Ibrahim J

    2017-01-01

    Human pluripotent stem-cell derived cardiomyocytes (hPSC-CMs) hold great promise for applications in human disease modeling, drug discovery, cardiotoxicity screening, and, ultimately, regenerative medicine. The ability to study multiple parameters of hPSC-CM function, such as contractile and

  19. Application of stem cell-derived retinal pigmented epithelium in retinal degenerative diseases: present and future

    Directory of Open Access Journals (Sweden)

    Mingyue Luo

    2018-01-01

    Full Text Available As a constituent of blood-retinal barrier and retinal outer segment (ROS scavenger, retinal pigmented epithelium (RPE is fundamental to normal function of retina. Malfunctioning of RPE contributes to the onset and advance of retinal degenerative diseases. Up to date, RPE replacement therapy is the only possible method to completely reverse retinal degeneration. Transplantation of human RPE stem cell-derived RPE (hRPESC-RPE has shown some good results in animal models. With promising results in terms of safety and visual improvement, human embryonic stem cell-derived RPE (hESC-RPE can be expected in clinical settings in the near future. Despite twists and turns, induced pluripotent stem cell-derived RPE (iPSC-RPE is now being intensely investigated to overcome genetic and epigenetic instability. By far, only one patient has received iPSC-RPE transplant, which is a hallmark of iPSC technology development. During follow-up, no major complications such as immunogenicity or tumorigenesis have been observed. Future trials should keep focusing on the safety of stem cell-derived RPE (SC-RPE especially in long period, and better understanding of the nature of stem cell and the molecular events in the process to generate SC-RPE is necessary to the prosperity of SC-RPE clinical application.

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

    Science.gov (United States)

    The establishment and initial characterization of a pig embryonic stem cell-derived pancreatic cell line, PICM-31, and a colony-cloned derivative cell line, PICM-31A, is described. The cell lines were propagated for several months at split ratios of 1:3 or 1:5 at each passage on STO feeder cells af...

  1. Bioinspired Cell-Derived Nanovesicles versus Exosomes as Drug Delivery Systems : A Cost-Effective Alternative

    NARCIS (Netherlands)

    Goh, Wei Jiang; Zou, Shui; Ong, Wei Yi; Torta, Federico; Alexandra, Alvarez Fernandez; Schiffelers, Raymond M.; Storm, Gert; Wang, Jiong-Wei; Czarny, Bertrand M S; Pastorin, Giorgia

    2017-01-01

    Cell Derived Nanovesicles (CDNs) have been developed from the rapidly expanding field of exosomes, representing a class of bioinspired Drug Delivery Systems (DDS). However, translation to clinical applications is limited by the low yield and multi-step approach in isolating naturally secreted

  2. Bioinspired Cell-Derived Nanovesicles versus Exosomes as Drug Delivery Systems : a Cost-Effective Alternative

    NARCIS (Netherlands)

    Goh, Wei Jiang; Zou, Shui; Ong, Wei Yi; Torta, Federico; Alexandra, Alvarez Fernandez; Schiffelers, Raymond M; Storm, G; Wang, Jiong Wei; Czarny, Bertrand; Pastorin, Giorgia

    2017-01-01

    Cell Derived Nanovesicles (CDNs) have been developed from the rapidly expanding field of exosomes, representing a class of bioinspired Drug Delivery Systems (DDS). However, translation to clinical applications is limited by the low yield and multi-step approach in isolating naturally secreted

  3. Application of stem cell-derived retinal pigmented epithelium in retinal degenerative diseases: present and future.

    Science.gov (United States)

    Luo, Mingyue; Chen, Youxin

    2018-01-01

    As a constituent of blood-retinal barrier and retinal outer segment (ROS) scavenger, retinal pigmented epithelium (RPE) is fundamental to normal function of retina. Malfunctioning of RPE contributes to the onset and advance of retinal degenerative diseases. Up to date, RPE replacement therapy is the only possible method to completely reverse retinal degeneration. Transplantation of human RPE stem cell-derived RPE (hRPESC-RPE) has shown some good results in animal models. With promising results in terms of safety and visual improvement, human embryonic stem cell-derived RPE (hESC-RPE) can be expected in clinical settings in the near future. Despite twists and turns, induced pluripotent stem cell-derived RPE (iPSC-RPE) is now being intensely investigated to overcome genetic and epigenetic instability. By far, only one patient has received iPSC-RPE transplant, which is a hallmark of iPSC technology development. During follow-up, no major complications such as immunogenicity or tumorigenesis have been observed. Future trials should keep focusing on the safety of stem cell-derived RPE (SC-RPE) especially in long period, and better understanding of the nature of stem cell and the molecular events in the process to generate SC-RPE is necessary to the prosperity of SC-RPE clinical application.

  4. Tumor cell-derived microparticles: a new form of cancer vaccine.

    Science.gov (United States)

    Zhang, Huafeng; Huang, Bo

    2015-08-01

    For cancer vaccines, tumor antigen availability is currently not an issue due to technical advances. However, the generation of optimal immune stimulation during vaccination is challenging. We have recently demonstrated that tumor cell-derived microparticles (MP) can function as a new form of potent cancer vaccine by efficiently activating type I interferon pathway in a cGAS/STING dependent manner.

  5. Analysis of Cell-Derived Microparticles with Highly Precise Nanotechnological Methods

    DEFF Research Database (Denmark)

    Cherré, Solène; Østergaard, Ole; Heegaard, Niels H.H.

    2014-01-01

    Cell-derived microparticles have gained a broad interest in the past years. Being released by blood cells upon activation or induction of apoptosis, they have a great potential as novel diagnostic markers and their investigation can bring new knowledge into the pathogenesis of various diseases. H...

  6. Integration of silicon-based neural probes and micro-drive arrays for chronic recording of large populations of neurons in behaving animals.

    Science.gov (United States)

    Michon, Frédéric; Aarts, Arno; Holzhammer, Tobias; Ruther, Patrick; Borghs, Gustaaf; McNaughton, Bruce; Kloosterman, Fabian

    2016-08-01

    Understanding how neuronal assemblies underlie cognitive function is a fundamental question in system neuroscience. It poses the technical challenge to monitor the activity of populations of neurons, potentially widely separated, in relation to behaviour. In this paper, we present a new system which aims at simultaneously recording from a large population of neurons from multiple separated brain regions in freely behaving animals. The concept of the new device is to combine the benefits of two existing electrophysiological techniques, i.e. the flexibility and modularity of micro-drive arrays and the high sampling ability of electrode-dense silicon probes. Newly engineered long bendable silicon probes were integrated into a micro-drive array. The resulting device can carry up to 16 independently movable silicon probes, each carrying 16 recording sites. Populations of neurons were recorded simultaneously in multiple cortical and/or hippocampal sites in two freely behaving implanted rats. Current approaches to monitor neuronal activity either allow to flexibly record from multiple widely separated brain regions (micro-drive arrays) but with a limited sampling density or to provide denser sampling at the expense of a flexible placement in multiple brain regions (neural probes). By combining these two approaches and their benefits, we present an alternative solution for flexible and simultaneous recordings from widely distributed populations of neurons in freely behaving rats.

  7. Altered calcium handling and increased contraction force in human embryonic stem cell derived cardiomyocytes following short term dexamethasone exposure

    NARCIS (Netherlands)

    Kosmidis, Georgios; Bellin, Milena; Ribeiro, Marcelo C.; van Meer, Berend; Ward-van Oostwaard, Dorien; Passier, Robert; Tertoolen, Leon G. J.; Mummery, Christine L.; Casini, Simona

    2015-01-01

    One limitation in using human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) for disease modeling and cardiac safety pharmacology is their immature functional phenotype compared with adult cardiomyocytes. Here, we report that treatment of human embryonic stem cell derived cardiomyocytes

  8. Single-Cell Transcriptomics Reveals a Population of Dormant Neural Stem Cells that Become Activated upon Brain Injury.

    Science.gov (United States)

    Llorens-Bobadilla, Enric; Zhao, Sheng; Baser, Avni; Saiz-Castro, Gonzalo; Zwadlo, Klara; Martin-Villalba, Ana

    2015-09-03

    Heterogeneous pools of adult neural stem cells (NSCs) contribute to brain maintenance and regeneration after injury. The balance of NSC activation and quiescence, as well as the induction of lineage-specific transcription factors, may contribute to diversity of neuronal and glial fates. To identify molecular hallmarks governing these characteristics, we performed single-cell sequencing of an unbiased pool of adult subventricular zone NSCs. This analysis identified a discrete, dormant NSC subpopulation that already expresses distinct combinations of lineage-specific transcription factors during homeostasis. Dormant NSCs enter a primed-quiescent state before activation, which is accompanied by downregulation of glycolytic metabolism, Notch, and BMP signaling and a concomitant upregulation of lineage-specific transcription factors and protein synthesis. In response to brain ischemia, interferon gamma signaling induces dormant NSC subpopulations to enter the primed-quiescent state. This study unveils general principles underlying NSC activation and lineage priming and opens potential avenues for regenerative medicine in the brain. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Near scale-free dynamics in neural population activity of waking/sleeping rats revealed by multiscale analysis.

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    Leonid A Safonov

    Full Text Available A neuron embedded in an intact brain, unlike an isolated neuron, participates in network activity at various spatial resolutions. Such multiple scale spatial dynamics is potentially reflected in multiple time scales of temporal dynamics. We identify such multiple dynamical time scales of the inter-spike interval (ISI fluctuations of neurons of waking/sleeping rats by means of multiscale analysis. The time scale of large non-Gaussianity in the ISI fluctuations, measured with the Castaing method, ranges up to several minutes, markedly escaping the low-pass filtering characteristics of neurons. A comparison between neural activity during waking and sleeping reveals that non-Gaussianity is stronger during waking than sleeping throughout the entire range of scales observed. We find a remarkable property of near scale independence of the magnitude correlations as the primary cause of persistent non-Gaussianity. Such scale-invariance of correlations is characteristic of multiplicative cascade processes and raises the possibility of the existence of a scale independent memory preserving mechanism.

  10. Involvement of multiple myeloma cell-derived exosomes in osteoclast differentiation

    OpenAIRE

    Raimondi, Lavinia; De Luca, Angela; Amodio, Nicola; Manno, Mauro; Raccosta, Samuele; Taverna, Simona; Bellavia, Daniele; Naselli, Flores; Fontana, Simona; Schillaci, Odessa; Giardino, Roberto; Fini, Milena; Tassone, Pierfrancesco; Santoro, Alessandra; De Leo, Giacomo

    2015-01-01

    Bone disease is the most frequent complication in multiple myeloma (MM) resulting in osteolytic lesions, bone pain, hypercalcemia and renal failure. In MM bone disease the perfect balance between bone-resorbing osteoclasts (OCs) and bone-forming osteoblasts (OBs) activity is lost in favour of OCs, thus resulting in skeletal disorders. Since exosomes have been described for their functional role in cancer progression, we here investigate whether MM cell-derived exosomes may be involved in OCs ...

  11. Potential of cancer cell-derived exosomes in clinical application: a review of recent research advances.

    Science.gov (United States)

    Sun, Yu; Liu, Jing

    2014-06-01

    Exosomes are 30- to 100-nm, membrane-bound vesicles that are released by most types of cells, including tumor cells. Exosomes contain a great variety of bioactive molecules, including signal peptides, microRNA, lipids, and DNA. In cancer, tumor cells aberrantly secrete large quantities of exosomes to transport paracrine signals or to contribute to tumor-environment interaction at a distance. The goal of this review was to discuss the recent advances on the mechanism of cancer-derived exosomes in tumor regulation. Pertinent articles and abstracts were identified through searches of PubMed for literature published from 1983 to December 2013. Search terms included exosome, tumor, cancer, diagnosis, and therapy. All of the exposed evidence points to communication between cancer cells and their surroundings, either mediated by cancer cell-derived exosomes or by stromal cell-derived exosomes. This communication probably supports tumor proliferation, motility, invasion, angiogenesis, and premetastatic niche preparation. In addition, recent research implies that cancer cell-derived exosomes play a suppressive role in cancer-directed immune response. The biomarkers detected in bodily fluid-derived exosomes imply a potential for exosomes in cancer diagnosis. Also, exosomes could be used as a vehicle to selectively deliver therapeutic nucleic-acid drugs or conventional drugs for tumor therapy. The tolerability and feasibility of cancer exosomes in diagnosis and therapy need to be further evaluated. Copyright © 2014 Elsevier HS Journals, Inc. All rights reserved.

  12. Functional neuromuscular junctions formed by embryonic stem cell-derived motor neurons.

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    Joy A Umbach

    Full Text Available A key objective of stem cell biology is to create physiologically relevant cells suitable for modeling disease pathologies in vitro. Much progress towards this goal has been made in the area of motor neuron (MN disease through the development of methods to direct spinal MN formation from both embryonic and induced pluripotent stem cells. Previous studies have characterized these neurons with respect to their molecular and intrinsic functional properties. However, the synaptic activity of stem cell-derived MNs remains less well defined. In this study, we report the development of low-density co-culture conditions that encourage the formation of active neuromuscular synapses between stem cell-derived MNs and muscle cells in vitro. Fluorescence microscopy reveals the expression of numerous synaptic proteins at these contacts, while dual patch clamp recording detects both spontaneous and multi-quantal evoked synaptic responses similar to those observed in vivo. Together, these findings demonstrate that stem cell-derived MNs innervate muscle cells in a functionally relevant manner. This dual recording approach further offers a sensitive and quantitative assay platform to probe disorders of synaptic dysfunction associated with MN disease.

  13. Plasma folate levels and associated factors in women planning to become pregnant in a population with high prevalence of neural tube defects.

    Science.gov (United States)

    Ma, Rui; Wang, Linlin; Jin, Lei; Li, Zhiwen; Ren, Aiguo

    2017-07-17

    Optimal blood folate levels of women before pregnancy are critical to the prevention of neural tube defects (NTDs). However, few studies have focused on blood folate levels of women planning to become pregnant. The aims of this study were to assess plasma folate levels in women who planned to become pregnant in a population with high prevalence of NTDs, to identify factors associated with plasma folate levels, and to evaluate the risk of NTDs at the population level. A total of 2065 women were enrolled at the time of premarital health check-up in two rural counties in northern China from November 2009 to December 2012. Fasting venous blood samples were collected and plasma folate concentrations were measured by microbiological method. The overall median of plasma folate was 10.5 nmol/L. 50% of the women had a plasma folate level below 10.5 nmol/L, a cutoff for megaloblastic anemia, and 88% below 18 nmol/L, a proposed optimal plasma folate level for the prevention of NTDs. Folic acid supplementation was the only factor to be associated with plasma folate concentrations, but only 1.9% of the women reported having taken folic acid supplements. A population risk of 29.3 NTD cases per 10,000 births was predicted. Women who planned to become pregnant had very low plasma folate in the population. Folic acid supplementation was the only factor to be associated with a high plasma folate concentration. High NTD risk would remain if women would get pregnant without having taken folic acid supplements. Birth Defects Research 109:1039-1047, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  14. Platelet-derived stromal cell-derived factor-1 is required for the transformation of circulating monocytes into multipotential cells.

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    Noriyuki Seta

    Full Text Available BACKGROUND: We previously described a primitive cell population derived from human circulating CD14(+ monocytes, named monocyte-derived multipotential cells (MOMCs, which are capable of differentiating into mesenchymal and endothelial lineages. To generate MOMCs in vitro, monocytes are required to bind to fibronectin and be exposed to soluble factor(s derived from circulating CD14(- cells. The present study was conducted to identify factors that induce MOMC differentiation. METHODS: We cultured CD14(+ monocytes on fibronectin in the presence or absence of platelets, CD14(- peripheral blood mononuclear cells, platelet-conditioned medium, or candidate MOMC differentiation factors. The transformation of monocytes into MOMCs was assessed by the presence of spindle-shaped adherent cells, CD34 expression, and the potential to differentiate in vitro into mesenchymal and endothelial lineages. RESULTS: The presence of platelets or platelet-conditioned medium was required to generate MOMCs from monocytes. A screening of candidate platelet-derived soluble factors identified stromal cell-derived factor (SDF-1 as a requirement for generating MOMCs. Blocking an interaction between SDF-1 and its receptor CXCR4 inhibited MOMC generation, further confirming SDF-1's critical role in this process. Finally, circulating MOMC precursors were found to reside in the CD14(+CXCR4(high cell population. CONCLUSION: The interaction of SDF-1 with CXCR4 is essential for the transformation of circulating monocytes into MOMCs.

  15. Muscle-derived stem cells isolated as non-adherent population give rise to cardiac, skeletal muscle and neural lineages.

    Science.gov (United States)

    Arsic, Nikola; Mamaeva, Daria; Lamb, Ned J; Fernandez, Anne

    2008-04-01

    Stem cells with the ability to differentiate in specialized cell types can be extracted from a wide array of adult tissues including skeletal muscle. Here we have analyzed a population of cells isolated from skeletal muscle on the basis of their poor adherence on uncoated or collagen-coated dishes that show multi-lineage differentiation in vitro. When analysed under proliferative conditions, these cells express stem cell surface markers Sca-1 (65%) and Bcrp-1 (80%) but also MyoD (15%), Neuronal beta III-tubulin (25%), GFAP (30%) or Nkx2.5 (1%). Although capable of growing as non-attached spheres for months, when given an appropriate matrix, these cells adhere giving rise to skeletal muscle, neuronal and cardiac muscle cell lineages. A similar cell population could not be isolated from either bone marrow or cardiac tissue suggesting their specificity to skeletal muscle. When injected into damaged muscle, these non-adherent muscle-derived cells are retrieved expressing Pax7, in a sublaminar position characterizing satellite cells and participate in forming new myofibers. These data show that a non-adherent stem cell population can be specifically isolated and expanded from skeletal muscle and upon attachment to a matrix spontaneously differentiate into muscle, cardiac and neuronal lineages in vitro. Although competing with resident satellite cells, these cells are shown to significantly contribute to repair of injured muscle in vivo supporting that a similar muscle-derived non-adherent cell population from human muscle may be useful in treatment of neuromuscular disorders.

  16. Muscle-derived stem cells isolated as non-adherent population give rise to cardiac, skeletal muscle and neural lineages

    International Nuclear Information System (INIS)

    Arsic, Nikola; Mamaeva, Daria; Lamb, Ned J.; Fernandez, Anne

    2008-01-01

    Stem cells with the ability to differentiate in specialized cell types can be extracted from a wide array of adult tissues including skeletal muscle. Here we have analyzed a population of cells isolated from skeletal muscle on the basis of their poor adherence on uncoated or collagen-coated dishes that show multi-lineage differentiation in vitro. When analysed under proliferative conditions, these cells express stem cell surface markers Sca-1 (65%) and Bcrp-1 (80%) but also MyoD (15%), Neuronal β III-tubulin (25%), GFAP (30%) or Nkx2.5 (1%). Although capable of growing as non-attached spheres for months, when given an appropriate matrix, these cells adhere giving rise to skeletal muscle, neuronal and cardiac muscle cell lineages. A similar cell population could not be isolated from either bone marrow or cardiac tissue suggesting their specificity to skeletal muscle. When injected into damaged muscle, these non-adherent muscle-derived cells are retrieved expressing Pax7, in a sublaminar position characterizing satellite cells and participate in forming new myofibers. These data show that a non-adherent stem cell population can be specifically isolated and expanded from skeletal muscle and upon attachment to a matrix spontaneously differentiate into muscle, cardiac and neuronal lineages in vitro. Although competing with resident satellite cells, these cells are shown to significantly contribute to repair of injured muscle in vivo supporting that a similar muscle-derived non-adherent cell population from human muscle may be useful in treatment of neuromuscular disorders

  17. Cell survival and differentiation with nanocrystalline glass-like carbon using substantia nigra dopaminergic cells derived from transgenic mouse embryos.

    Directory of Open Access Journals (Sweden)

    Noela Rodriguez-Losada

    Full Text Available Regenerative medicine requires, in many cases, physical supports to facilitate appropriate cellular architecture, cell polarization and the improvement of the correct differentiation processes of embryonic stem cells, induced pluripotent cells or adult cells. Because the interest in carbon nanomaterials has grown within the last decade in light of a wide variety of applications, the aim of this study was to test and evaluate the suitability and cytocompatibility of a particular nanometer-thin nanocrystalline glass-like carbon film (NGLC composed of curved graphene flakes joined by an amorphous carbon matrix. This material is a disordered structure with high transparency and electrical conductivity. For this purpose, we used a cell line (SN4741 from substantia nigra dopaminergic cells derived from transgenic mouse embryos. Cells were cultured either in a powder of increasing concentrations of NGLC microflakes (82±37μm in the medium or on top of nanometer-thin films bathed in the same culture medium. The metabolism activity of SN4741 cells in presence of NGLC was assessed using methylthiazolyldiphenyl-tetrazolium (MTT and apoptosis/necrosis flow cytometry assay respectively. Growth and proliferation as well as senescence were demonstrated by western blot (WB of proliferating cell nuclear antigen (PCNA, monoclonal phosphorylate Histone 3 (serine 10 (PH3 and SMP30 marker. Specific dopaminergic differentiation was confirmed by the WB analysis of tyrosine hydroxylase (TH. Cell maturation and neural capability were characterized using specific markers (SYP: synaptophysin and GIRK2: G-protein-regulated inward-rectifier potassium channel 2 protein via immunofluorescence and coexistence measurements. The results demonstrated cell positive biocompatibility with different concentrations of NGLC. The cells underwent a process of adaptation of SN4741 cells to NGLC where their metabolism decreases. This process is related to a decrease of PH3 expression and

  18. [Characterization of stem cells derived from the neonatal auditory sensory epithelium].

    Science.gov (United States)

    Diensthuber, M; Heller, S

    2010-11-01

    In contrast to regenerating hair cell-bearing organs of nonmammalian vertebrates the adult mammalian organ of Corti appears to have lost its ability to maintain stem cells. The result is a lack of regenerative ability and irreversible hearing loss following auditory hair cell death. Unexpectedly, the neonatal auditory sensory epithelium has recently been shown to harbor cells with stem cell features. The origin of these cells within the cochlea's sensory epithelium is unknown. We applied a modified neurosphere assay to identify stem cells within distinct subregions of the neonatal mouse auditory sensory epithelium. Sphere cells were characterized by multiple markers and morphologic techniques. Our data reveal that both the greater and the lesser epithelial ridge contribute to the sphere-forming stem cell population derived from the auditory sensory epithelium. These self-renewing sphere cells express a variety of markers for neural and otic progenitor cells and mature inner ear cell types. Stem cells can be isolated from specific regions of the auditory sensory epithelium. The distinct features of these cells imply a potential application in the development of a cell replacement therapy to regenerate the damaged sensory epithelium.

  19. A subpopulation of smooth muscle cells, derived from melanocyte-competent precursors, prevents patent ductus arteriosus.

    Directory of Open Access Journals (Sweden)

    Ichiro Yajima

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

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

    Science.gov (United States)

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

    2013-01-01

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

  1. Characterizing human stem cell-derived sensory neurons at the single-cell level reveals their ion channel expression and utility in pain research.

    Science.gov (United States)

    Young, Gareth T; Gutteridge, Alex; Fox, Heather DE; Wilbrey, Anna L; Cao, Lishuang; Cho, Lily T; Brown, Adam R; Benn, Caroline L; Kammonen, Laura R; Friedman, Julia H; Bictash, Magda; Whiting, Paul; Bilsland, James G; Stevens, Edward B

    2014-08-01

    The generation of human sensory neurons by directed differentiation of pluripotent stem cells opens new opportunities for investigating the biology of pain. The inability to generate this cell type has meant that up until now their study has been reliant on the use of rodent models. Here, we use a combination of population and single-cell techniques to perform a detailed molecular, electrophysiological, and pharmacological phenotyping of sensory neurons derived from human embryonic stem cells. We describe the evolution of cell populations over 6 weeks of directed differentiation; a process that results in the generation of a largely homogeneous population of neurons that are both molecularly and functionally comparable to human sensory neurons derived from mature dorsal root ganglia. This work opens the prospect of using pluripotent stem-cell-derived sensory neurons to study human neuronal physiology and as in vitro models for drug discovery in pain and sensory disorders.

  2. Differentiation and Application of Induced Pluripotent Stem Cell-Derived Vascular Smooth Muscle Cells.

    Science.gov (United States)

    Maguire, Eithne Margaret; Xiao, Qingzhong; Xu, Qingbo

    2017-11-01

    Vascular smooth muscle cells (VSMCs) play a role in the development of vascular disease, for example, neointimal formation, arterial aneurysm, and Marfan syndrome caused by genetic mutations in VSMCs, but little is known about the mechanisms of the disease process. Advances in induced pluripotent stem cell technology have now made it possible to derive VSMCs from several different somatic cells using a selection of protocols. As such, researchers have set out to delineate key signaling processes involved in triggering VSMC gene expression to grasp the extent of gene regulatory networks involved in phenotype commitment. This technology has also paved the way for investigations into diseases affecting VSMC behavior and function, which may be treatable once an identifiable culprit molecule or gene has been repaired. Moreover, induced pluripotent stem cell-derived VSMCs are also being considered for their use in tissue-engineered blood vessels as they may prove more beneficial than using autologous vessels. Finally, while several issues remains to be clarified before induced pluripotent stem cell-derived VSMCs can become used in regenerative medicine, they do offer both clinicians and researchers hope for both treating and understanding vascular disease. In this review, we aim to update the recent progress on VSMC generation from stem cells and the underlying molecular mechanisms of VSMC differentiation. We will also explore how the use of induced pluripotent stem cell-derived VSMCs has changed the game for regenerative medicine by offering new therapeutic avenues to clinicians, as well as providing researchers with a new platform for modeling of vascular disease. © 2017 American Heart Association, Inc.

  3. Association of mast cell-derived VEGF and proteases in Dengue shock syndrome.

    Directory of Open Access Journals (Sweden)

    Takahisa Furuta

    Full Text Available BACKGROUND: Recent in-vitro studies have suggested that mast cells are involved in Dengue virus infection. To clarify the role of mast cells in the development of clinical Dengue fever, we compared the plasma levels of several mast cell-derived mediators (vascular endothelial cell growth factor [VEGF], soluble VEGF receptors [sVEGFRs], tryptase, and chymase and -related cytokines (IL-4, -9, and -17 between patients with differing severity of Dengue fever and healthy controls. METHODOLOGY/PRINCIPAL FINDINGS: The study was performed at Children's Hospital No. 2, Ho Chi Minh City, and Vinh Long Province Hospital, Vietnam from 2002 to 2005. Study patients included 103 with Dengue fever (DF, Dengue hemorrhagic fever (DHF, and Dengue shock syndrome (DSS, as diagnosed by the World Health Organization criteria. There were 189 healthy subjects, and 19 febrile illness patients of the same Kinh ethnicity. The levels of mast cell-derived mediators and -related cytokines in plasma were measured by ELISA. VEGF and sVEGFR-1 levels were significantly increased in DHF and DSS compared with those of DF and controls, whereas sVEGFR-2 levels were significantly decreased in DHF and DSS. Significant increases in tryptase and chymase levels, which were accompanied by high IL-9 and -17 concentrations, were detected in DHF and DSS patients. By day 4 of admission, VEGF, sVEGFRs, and proteases levels had returned to similar levels as DF and controls. In-vitro VEGF production by mast cells was examined in KU812 and HMC-1 cells, and was found to be highest when the cells were inoculated with Dengue virus and human Dengue virus-immune serum in the presence of IL-9. CONCLUSIONS: As mast cells are an important source of VEGF, tryptase, and chymase, our findings suggest that mast cell activation and mast cell-derived mediators participate in the development of DHF. The two proteases, particularly chymase, might serve as good predictive markers of Dengue disease severity.

  4. Circulating cell-derived microparticles in patients with minimally symptomatic obstructive sleep apnoea.

    Science.gov (United States)

    Ayers, L; Ferry, B; Craig, S; Nicoll, D; Stradling, J R; Kohler, M

    2009-03-01

    Moderate-severe obstructive sleep apnoea (OSA) has been associated with several pro-atherogenic mechanisms and increased cardiovascular risk, but it is not known if minimally symptomatic OSA has similar effects. Circulating cell-derived microparticles have been shown to have pro-inflammatory, pro-coagulant and endothelial function-impairing effects, as well as to predict subclinical atherosclerosis and cardiovascular risk. In 57 patients with minimally symptomatic OSA, and 15 closely matched control subjects without OSA, AnnexinV-positive, platelet-, leukocyte- and endothelial cell-derived microparticles were measured by flow cytometry. In patients with OSA, median (interquartile range) levels of AnnexinV-positive microparticles were significantly elevated compared with control subjects: 2,586 (1,566-3,964) microL(-1) versus 1,206 (474-2,501) microL(-1), respectively. Levels of platelet-derived and leukocyte-derived microparticles were also significantly higher in patients with OSA (2,267 (1,102-3,592) microL(-1) and 20 (14-31) microL(-1), respectively) compared with control subjects (925 (328-2,068) microL(-1) and 15 (5-23) microL(-1), respectively). Endothelial cell-derived microparticle levels were similar in patients with OSA compared with control subjects (13 (8-25) microL(-1) versus 11 (6-17) microL(-1)). In patients with minimally symptomatic obstructive sleep apnoea, levels of AnnexinV-positive, platelet- and leukocyte-derived microparticles are elevated when compared with closely matched control subjects without obstructive sleep apnoea. These findings suggest that these patients may be at increased cardiovascular risk, despite being minimally symptomatic.

  5. Characterization of calcium signals in human induced pluripotent stem cell-derived dentate gyrus neuronal progenitors and mature neurons, stably expressing an advanced calcium indicator protein.

    Science.gov (United States)

    Vőfély, Gergő; Berecz, Tünde; Szabó, Eszter; Szebényi, Kornélia; Hathy, Edit; Orbán, Tamás I; Sarkadi, Balázs; Homolya, László; Marchetto, Maria C; Réthelyi, János M; Apáti, Ágota

    2018-04-01

    Pluripotent stem cell derived human neuronal progenitor cells (hPSC-NPCs) and their mature neuronal cell culture derivatives may efficiently be used for central nervous system (CNS) drug screening, including the investigation of ligand-induced calcium signalization. We have established hippocampal NPC cultures derived from human induced PSCs, which were previously generated by non-integrating Sendai virus reprogramming. Using established protocols these NPCs were differentiated into hippocampal dentate gyrus neurons. In order to study calcium signaling without the need of dye loading, we have stably expressed an advanced calcium indicator protein (GCaMP6fast) in the NPCs using the Sleeping Beauty transposon system. We observed no significant effects of the long-term GCaMP6 expression on NPC morphology, gene expression pattern or neural differentiation capacity. In order to compare the functional properties of GCaMP6-expressing neural cells and the corresponding parental cells loaded with calcium indicator dye Fluo-4, a detailed characterization of calcium signals was performed. We found that the calcium signals induced by ATP, glutamate, LPA, or proteases - were similar in these two systems. Moreover, the presence of the calcium indicator protein allowed for a sensitive, repeatable detection of changes in calcium signaling during the process of neurogenesis and neuronal maturation. Copyright © 2018 Elsevier Inc. All rights reserved.

  6. Drug-loaded nanoparticles induce gene expression in human pluripotent stem cell derivatives

    Science.gov (United States)

    Gajbhiye, Virendra; Escalante, Leah; Chen, Guojun; Laperle, Alex; Zheng, Qifeng; Steyer, Benjamin; Gong, Shaoqin; Saha, Krishanu

    2013-12-01

    Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained higher fibroblast proliferation levels and MMP activity. The results demonstrate that the PEG-H40-DXC nanoparticle system provides an effective tool to controlling gene expression in human stem cell derivatives.Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained

  7. Activation of different neural precursor populations in the adult hippocampus: does this lead to new neurons with discrete functions?

    Science.gov (United States)

    Jhaveri, Dhanisha J; Taylor, Chanel J; Bartlett, Perry F

    2012-07-01

    Resident populations of stem and precursor cells drive the production of new neurons in the adult hippocampus. Recent discoveries have highlighted that a large proportion of these precursor cells are in fact quiescent and can be activated by distinct neuronal activity under both normal physiological and pathological conditions. As growing evidence indicates that newborn neurons play a critical role in cognitive functions such as learning and memory and in mood regulation, it is paramount that we obtain a better understanding of how the reservoirs of stem and precursor cells are maintained and activated. In this review, we critically examine the roles of key molecular mechanisms that have been shown to regulate hippocampal precursor cells, especially their activation. We believe that understanding the mechanistic details of the activity-driven regulation of precursor cells will equip us with the ability to develop tailored strategies to trigger the generation of new neurons, thereby improving the functional outcomes in various neurological and psychiatric conditions. Copyright © 2012 Wiley Periodicals, Inc.

  8. Comparing deep neural network and other machine learning algorithms for stroke prediction in a large-scale population-based electronic medical claims database.

    Science.gov (United States)

    Chen-Ying Hung; Wei-Chen Chen; Po-Tsun Lai; Ching-Heng Lin; Chi-Chun Lee

    2017-07-01

    Electronic medical claims (EMCs) can be used to accurately predict the occurrence of a variety of diseases, which can contribute to precise medical interventions. While there is a growing interest in the application of machine learning (ML) techniques to address clinical problems, the use of deep-learning in healthcare have just gained attention recently. Deep learning, such as deep neural network (DNN), has achieved impressive results in the areas of speech recognition, computer vision, and natural language processing in recent years. However, deep learning is often difficult to comprehend due to the complexities in its framework. Furthermore, this method has not yet been demonstrated to achieve a better performance comparing to other conventional ML algorithms in disease prediction tasks using EMCs. In this study, we utilize a large population-based EMC database of around 800,000 patients to compare DNN with three other ML approaches for predicting 5-year stroke occurrence. The result shows that DNN and gradient boosting decision tree (GBDT) can result in similarly high prediction accuracies that are better compared to logistic regression (LR) and support vector machine (SVM) approaches. Meanwhile, DNN achieves optimal results by using lesser amounts of patient data when comparing to GBDT method.

  9. Scalable Electrophysiological Investigation of iPS Cell-Derived Cardiomyocytes Obtained by a Lentiviral Purification Strategy

    Directory of Open Access Journals (Sweden)

    Stephanie Friedrichs

    2015-01-01

    Full Text Available Disease-specific induced pluripotent stem (iPS cells can be generated from patients and differentiated into functional cardiomyocytes for characterization of the disease and for drug screening. In order to obtain pure cardiomyocytes for automated electrophysiological investigation, we here report a novel non-clonal purification strategy by using lentiviral gene transfer of a puromycin resistance gene under the control of a cardiac-specific promoter. We have applied this method to our previous reported wild-type and long QT syndrome 3 (LQTS 3-specific mouse iPS cells and obtained a pure cardiomyocyte population. These cells were investigated by action potential analysis with manual and automatic planar patch clamp technologies, as well as by recording extracellular field potentials using a microelectrode array system. Action potentials and field potentials showed the characteristic prolongation at low heart rates in LQTS 3-specific, but not in wild-type iPS cell-derived cardiomyocytes. Hence, LQTS 3-specific cardiomyocytes can be purified from iPS cells with a lentiviral strategy, maintain the hallmarks of the LQTS 3 disease and can be used for automated electrophysiological characterization and drug screening.

  10. Evaluation of hollow fiber culture for large-scale production of mouse embryonic stem cell-derived hematopoietic stem cells.

    Science.gov (United States)

    Nakano, Yu; Iwanaga, Shinya; Mizumoto, Hiroshi; Kajiwara, Toshihisa

    2018-03-03

    Hematopoietic stem cells (HSCs) have the ability to differentiate into all types of blood cells and can be transplanted to treat blood disorders. However, it is difficult to obtain HSCs in large quantities because of the shortage of donors. Recent efforts have focused on acquiring HSCs by differentiation of pluripotent stem cells. As a conventional differentiation method of pluripotent stem cells, the formation of embryoid bodies (EBs) is often employed. However, the size of EBs is limited by depletion of oxygen and nutrients, which prevents them from being efficient for the production of HSCs. In this study, we developed a large-scale hematopoietic differentiation approach for mouse embryonic stem (ES) cells by applying a hollow fiber (HF)/organoid culture method. Cylindrical organoids, which had the potential for further spontaneous differentiation, were established inside of hollow fibers. Using this method, we improved the proliferation rate of mouse ES cells to produce an increased HSC population and achieved around a 40-fold higher production volume of HSCs in HF culture than in conventional EB culture. Therefore, the HF/organoid culture method may be a new mass culture method to acquire pluripotent stem cell-derived HSCs.

  11. Sympathetic neurons modulate the beat rate of pluripotent cell-derived cardiomyocytes in vitro.

    Science.gov (United States)

    Takeuchi, Akimasa; Shimba, Kenta; Mori, Masahide; Takayama, Yuzo; Moriguchi, Hiroyuki; Kotani, Kiyoshi; Lee, Jong-Kook; Noshiro, Makoto; Jimbo, Yasuhiko

    2012-12-01

    Although stem cell-derived cardiomyocytes have great potential for the therapy of heart failure, it is unclear whether their function after grafting can be controlled by the host sympathetic nervous system, a component of the autonomic nervous system (ANS). Here we demonstrate the formation of functional connections between rat sympathetic superior cervical ganglion (SCG) neurons and pluripotent (P19.CL6) cell-derived cardiomyocytes (P19CMs) in compartmentalized co-culture, achieved using photolithographic microfabrication techniques. Formation of synapses between sympathetic neurons and P19CMs was confirmed by immunostaining with antibodies against β-3 tubulin, synapsin I and cardiac troponin-I. Changes in the beat rate of P19CMs were triggered after electrical stimulation of the co-cultured SCG neurons, and were affected by the pulse frequency of the electrical stimulation. Such changes in the beat rate were prevented when propranolol, a β-adrenoreceptor antagonist, was added to the culture medium. These results suggest that the beat rate of differentiated cardiomyocytes can be modulated by electrical stimulation of connected sympathetic neurons.

  12. Data on importance of hematopoietic cell derived Lipocalin 2 against gut inflammation

    Directory of Open Access Journals (Sweden)

    Piu Saha

    2016-09-01

    Full Text Available The data herein is related to the research article entitled “Microbiota-inducible innate immune siderophore binding protein Lipocalin 2 is critical for intestinal homeostasis” (Singh et al., 2016 [1]. In the present article, we monitored dextran sodium sulfate (DSS-induced colitis development upon Lipocalin 2 (Lcn2 neutralization, and examined the survival of Lcn2 deficient (Lcn2KO mice and their WT littermates upon DSS challenge. To dissect the relative contribution of immune and non-immune cells-derived Lcn2 in mediating protection against gut inflammation, we generated respective bone marrow chimera and evaluated their susceptibility to IL-10 receptor neutralization-induced chronic colitis.Neutralization of Lcn2 in WT mice resulted in exacerbated DSS-induced colitis. Notably, mice lacking Lcn2 exhibited 100% mortality whereas only 20% mortality was observed in WT mice upon DSS challenge. Further, data from bone marrow chimera showed that immune cell-derived Lcn2 is the major contributor in conferring protection against colitis. Keywords: Siderocalin, Gut microbiota, Inflammatory bowel disease, Bone marrow chimeras, Colitis

  13. Decellularized Tissue and Cell-Derived Extracellular Matrices as Scaffolds for Orthopaedic Tissue Engineering

    Science.gov (United States)

    Cheng, Christina W.; Solorio, Loran D.; Alsberg, Eben

    2014-01-01

    The reconstruction of musculoskeletal defects is a constant challenge for orthopaedic surgeons. Musculoskeletal injuries such as fractures, chondral lesions, infections and tumor debulking can often lead to large tissue voids requiring reconstruction with tissue grafts. Autografts are currently the gold standard in orthopaedic tissue reconstruction; however, there is a limit to the amount of tissue that can be harvested before compromising the donor site. Tissue engineering strategies using allogeneic or xenogeneic decellularized bone, cartilage, skeletal muscle, tendon and ligament have emerged as promising potential alternative treatment. The extracellular matrix provides a natural scaffold for cell attachment, proliferation and differentiation. Decellularization of in vitro cell-derived matrices can also enable the generation of autologous constructs from tissue specific cells or progenitor cells. Although decellularized bone tissue is widely used clinically in orthopaedic applications, the exciting potential of decellularized cartilage, skeletal muscle, tendon and ligament cell-derived matrices has only recently begun to be explored for ultimate translation to the orthopaedic clinic. PMID:24417915

  14. G{sub 2} radiosensitivity of cells derived from cancer-prone individuals

    Energy Technology Data Exchange (ETDEWEB)

    Darroudi, F.; Vyas, R.C.; Vermeulen, S.; Natarajan, A.T. [J.A. Cohen Institute of Radiopathology and Radiation Protection, Interuniversity Institute, Leiden (Netherlands)

    1995-04-01

    The potential of enhanced chromatid damage, observed after X-irradiation of G{sub 2} phase, has been used to detect individuals genetically predisposed to cancer, utilising fibroblasts/lymphocytes from these patients as well as fibroblasts derived from human tumours. Fibroblasts and/or lymphocyte samples of two autosomal recessive syndromes (xeroderma pigmentosum (XP), Fanconi`s anaemia (FA)) and one congenital or acquired disorder, aplastic anaemia (AA), were employed for the G{sub 2} radiosensitivity assay. In addition, we have estimated the frequencies of spontaneously occurring chromosomal aberrations as well as G{sub 2} radiosensitivity of eight samples of fibroblasts/fibroblast-like cells (two normal, two colorectal carcinoma, two Wilms` tumour, one retinoblastoma and one polyposis coli), and three samples of lymphocytes (two normal and one from a lymphoma patient). The results obtained indicate that there were no differences between fibroblast cells derived from patients or tumours, except FA patients, in the frequency of spontaneously occurring chromosomal aberrations when compared to normal cells. Following X-irradiation we did not observe any significantly increased G{sub 2} radiosensitivity in FA and XP cells. Lymphocytes from AA and lymphoma patients, and all tumour cell lines except retinoblastoma, responded with increased frequencies of aberrations following G{sub 2} X-irradiation in comparison to cells derived from normal individuals. In our hands, the G{sub 2} sensitivity assay could not always discriminate cells from cancer-prone individuals from those of controls.

  15. Functions of Müller cell-derived vascular endothelial growthfactor in diabetic retinopathy

    Institute of Scientific and Technical Information of China (English)

    2015-01-01

    Müller cells are macroglia and play many essentialroles as supporting cells in the retina. To respond topathological changes in diabetic retinopathy (DR), amajor complication in the eye of diabetic patients,retinal Müller glia produce a high level of vascularendothelial growth factor (VEGF or VEGF-A). As VEGFis expressed by multiple retinal cell-types and Müllerglia comprise only a small portion of cells in the retina,it has been a great challenge to reveal the function ofVEGF or other globally expressed proteins produced byMüller cells. With the development of conditional genetargeting tools, it is now possible to dissect the functionof Müller cell-derived VEGF in vivo . By using conditionalgene targeting approach, we demonstrate that Müllerglia are a major source of retinal VEGF in diabetic miceand Müller cell-derived VEGF plays a significant role inthe alteration of protein expression and peroxynitration,which leads to retinal inflammation, neovascularization,vascular leakage, and vascular lesion, key pathologicalchanges in DR. Therefore, Müller glia are a potentialcellular target for the treatment of DR, a leading causeof blindness.

  16. Mesenchymal stromal cell derived endothelial progenitor treatment in patients with refractory angina

    DEFF Research Database (Denmark)

    Friis, Tina; Haack-Sørensen, Mandana; Mathiasen, Anders B

    2011-01-01

    Abstract Aims. We evaluated the feasibility, safety and efficacy of intra-myocardial injection of autologous mesenchymal stromal cells derived endothelial progenitor cell (MSC) in patients with stable coronary artery disease (CAD) and refractory angina in this first in man trial. Methods and resu......Abstract Aims. We evaluated the feasibility, safety and efficacy of intra-myocardial injection of autologous mesenchymal stromal cells derived endothelial progenitor cell (MSC) in patients with stable coronary artery disease (CAD) and refractory angina in this first in man trial. Methods...... and results. A total of 31 patients with stable CAD, moderate to severe angina and no further revascularization options, were included. Bone marrow MSC were isolated and culture expanded for 6-8 weeks. It was feasible and safe to establish in-hospital culture expansion of autologous MSC and perform intra......-myocardial injection of MSC. After six months follow-up myocardial perfusion was unaltered, but the patients increased exercise capacity (p angina attacks (p Angina Questionnaire (SAQ) evaluations (p

  17. Cell-derived microparticles and vascular pregnancy complications: a systematic and comprehensive review.

    Science.gov (United States)

    Alijotas-Reig, Jaume; Palacio-Garcia, Carles; Llurba, Elisa; Vilardell-Tarres, Miquel

    2013-02-01

    To assess current studies on the relationship between cell-derived microparticles (cMP) and recurrent miscarriages (RM) and pre-eclampsia (PE), and review the relationships between cMP and inflammatory and clot pathways, antiphospholipid antibodies (aPL), cytokines, and pregnancy complications. Systematic and comprehensive review of the literature from January 2000 to January 2012. Vall d'Hebron University Hospital. Women with recurrent miscarriages or PE, healthy nonpregnant women, and healthy pregnant women. None. Comparison of cMP numbers and types among groups. Platelet and endothelial cMP are increased in women with normal pregnancies compared with nonpregnant healthy women. Only five case-control studies regarding cMP and RM and 16 on cMP and PE were found to match our objective. Three of five articles referring to RM showed differences in cMP numbering, and 13 of 16 on cMP and PE showed differences in some type of cMP compared with controls. Cell-derived microparticles were raised in normal pregnancy. Recurrent miscarriage seems to be related to endothelial and platelet cell activation and/or consumption. An increase in almost all cMP types was observed in PE. A relationship between cMP and endothelial activation and proinflammatory status seems to exist. Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  18. A novel method to generate and culture human mast cells: Peripheral CD34+ stem cell-derived mast cells (PSCMCs).

    Science.gov (United States)

    Schmetzer, Oliver; Valentin, Patricia; Smorodchenko, Anna; Domenis, Rossana; Gri, Giorgia; Siebenhaar, Frank; Metz, Martin; Maurer, Marcus

    2014-11-01

    The identification and characterization of human mast cell (MC) functions are hindered by the shortage of MC populations suitable for investigation. Here, we present a novel technique for generating large numbers of well differentiated and functional human MCs from peripheral stem cells (=peripheral stem cell-derived MCs, PSCMCs). Innovative and key features of this technique include 1) the use of stem cell concentrates, which are routinely discarded by blood banks, as the source of CD34+ stem cells, 2) cell culture in serum-free medium and 3) the addition of LDL as well as selected cytokines. In contrast to established and published protocols that use CD34+ or CD133+ progenitor cells from full blood, we used a pre-enriched cell population obtained from stem cell concentrates, which yielded up to 10(8) differentiated human MCs per batch after only three weeks of culture starting with 10(6) total CD34+ cells. The total purity on MCs (CD117+, FcεR1+) generated by this method varied between 55 and 90%, of which 4-20% were mature MCs that contain tryptase and chymase and show expression of FcεRI and CD117 in immunohistochemistry. PSCMCs showed robust histamine release in response to stimulation with anti-FcεR1 or IgE/anti-IgE, and increased proliferation and differentiation in response to IL-1β or IFN-γ. Taken together, this new protocol of the generation of large numbers of human MCs provides for an innovative and suitable option to investigate the biology of human MCs. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Mesenchymal Stem/Progenitor Cells Derived from Articular Cartilage, Synovial Membrane and Synovial Fluid for Cartilage Regeneration: Current Status and Future Perspectives.

    Science.gov (United States)

    Huang, Yi-Zhou; Xie, Hui-Qi; Silini, Antonietta; Parolini, Ornella; Zhang, Yi; Deng, Li; Huang, Yong-Can

    2017-10-01

    Large articular cartilage defects remain an immense challenge in the field of regenerative medicine because of their poor intrinsic repair capacity. Currently, the available medical interventions can relieve clinical symptoms to some extent, but fail to repair the cartilaginous injuries with authentic hyaline cartilage. There has been a surge of interest in developing cell-based therapies, focused particularly on the use of mesenchymal stem/progenitor cells with or without scaffolds. Mesenchymal stem/progenitor cells are promising graft cells for tissue regeneration, but the most suitable source of cells for cartilage repair remains controversial. The tissue origin of mesenchymal stem/progenitor cells notably influences the biological properties and therapeutic potential. It is well known that mesenchymal stem/progenitor cells derived from synovial joint tissues exhibit superior chondrogenic ability compared with those derived from non-joint tissues; thus, these cell populations are considered ideal sources for cartilage regeneration. In addition to the progress in research and promising preclinical results, many important research questions must be answered before widespread success in cartilage regeneration is achieved. This review outlines the biology of stem/progenitor cells derived from the articular cartilage, the synovial membrane, and the synovial fluid, including their tissue distribution, function and biological characteristics. Furthermore, preclinical and clinical trials focusing on their applications for cartilage regeneration are summarized, and future research perspectives are discussed.

  20. Electrophysiological properties and calcium handling of embryonic stem cell-derived cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Jae Boum Youm

    2016-03-01

    Full Text Available Embryonic stem cell-derived cardiomyocytes (ESC-CMs hold great interest in many fields of research including clinical applications such as stem cell and gene therapy for cardiac repair or regeneration. ESC-CMs are also used as a platform tool for pharmacological tests or for investigations of cardiac remodeling. ESC-CMs have many different aspects of morphology, electrophysiology, calcium handling, and bioenergetics compared with adult cardiomyocytes. They are immature in morphology, similar to sinus nodal-like in the electrophysiology, higher contribution of trans-sarcolemmal Ca2+ influx to Ca2+ handling, and higher dependence on anaerobic glycolysis. Here, I review a detailed electrophysiology and Ca2+ handling features of ESC-CMs during differentiation into adult cardiomyocytes to gain insights into how all the developmental changes are related to each other to display cardinal features of developing cardiomyocytes.

  1. Generation of a Motor Nerve Organoid with Human Stem Cell-Derived Neurons

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    Jiro Kawada

    2017-11-01

    Full Text Available During development, axons spontaneously assemble into a fascicle to form nerves and tracts in the nervous system as they extend within a spatially constrained path. However, understanding of the axonal fascicle has been hampered by lack of an in vitro model system. Here, we report generation of a nerve organoid composed of a robust fascicle of axons extended from a spheroid of human stem cell-derived motor neurons within our custom-designed microdevice. The device is equipped with a narrow channel providing a microenvironment that facilitates the growing axons to spontaneously assemble into a unidirectional fascicle. The fascicle was specifically made with axons. We found that it was electrically active and elastic and could serve as a model to evaluate degeneration of axons in vitro. This nerve organoid model should facilitate future studies on the development of the axonal fascicle and drug screening for diseases affecting axon fascicles.

  2. Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Afford New Opportunities in Inherited Cardiovascular Disease Modeling

    Directory of Open Access Journals (Sweden)

    Daniel R. Bayzigitov

    2016-01-01

    Full Text Available Fundamental studies of molecular and cellular mechanisms of cardiovascular disease pathogenesis are required to create more effective and safer methods of their therapy. The studies can be carried out only when model systems that fully recapitulate pathological phenotype seen in patients are used. Application of laboratory animals for cardiovascular disease modeling is limited because of physiological differences with humans. Since discovery of induced pluripotency generating induced pluripotent stem cells has become a breakthrough technology in human disease modeling. In this review, we discuss a progress that has been made in modeling inherited arrhythmias and cardiomyopathies, studying molecular mechanisms of the diseases, and searching for and testing drug compounds using patient-specific induced pluripotent stem cell-derived cardiomyocytes.

  3. Induced Pluripotent Stem Cell-Derived Endothelial Cells in Insulin Resistance and Metabolic Syndrome.

    Science.gov (United States)

    Carcamo-Orive, Ivan; Huang, Ngan F; Quertermous, Thomas; Knowles, Joshua W

    2017-11-01

    Insulin resistance leads to a number of metabolic and cellular abnormalities including endothelial dysfunction that increase the risk of vascular disease. Although it has been particularly challenging to study the genetic determinants that predispose to abnormal function of the endothelium in insulin-resistant states, the possibility of deriving endothelial cells from induced pluripotent stem cells generated from individuals with detailed clinical phenotyping, including accurate measurements of insulin resistance accompanied by multilevel omic data (eg, genetic and genomic characterization), has opened new avenues to study this relationship. Unfortunately, several technical barriers have hampered these efforts. In the present review, we summarize the current status of induced pluripotent stem cell-derived endothelial cells for modeling endothelial dysfunction associated with insulin resistance and discuss the challenges to overcoming these limitations. © 2017 American Heart Association, Inc.

  4. DNA repair ability of cultured cells derived from mouse embryos in comparison with human cells

    International Nuclear Information System (INIS)

    Yaki, T.

    1982-01-01

    DNA repair in mouse cells derived from embryos of 3 inbred strains were investigated in comparison with that in human cells. The levels of unscheduled DNA synthesis after UV irradiation appeared to change at different passages, but capacities of host-cell reactivation of UV-irradiated herpes simplex virus were always reduced to the same levels as those in xeroderma pigmentosum cells. This implied that mouse cells are reduced in excision-repair capacities and that the apparently high levels of unscheduled DNA synthesis at certain passages are not quantitatively related to high levels of cell survival. Essentially no differences in DNA repair were noted among 3 strains - BALB/c, C3H/He and C57BL/10. (orig.)

  5. Neural networks

    International Nuclear Information System (INIS)

    Denby, Bruce; Lindsey, Clark; Lyons, Louis

    1992-01-01

    The 1980s saw a tremendous renewal of interest in 'neural' information processing systems, or 'artificial neural networks', among computer scientists and computational biologists studying cognition. Since then, the growth of interest in neural networks in high energy physics, fueled by the need for new information processing technologies for the next generation of high energy proton colliders, can only be described as explosive

  6. Chronic inhibition of tumor cell-derived VEGF enhances the malignant phenotype of colorectal cancer cells

    International Nuclear Information System (INIS)

    Yamagishi, Naoko; Teshima-Kondo, Shigetada; Masuda, Kiyoshi; Nishida, Kensei; Kuwano, Yuki; Dang, Duyen T; Dang, Long H; Nikawa, Takeshi; Rokutan, Kazuhito

    2013-01-01

    Vascular endothelial growth factor-a (VEGF)-targeted therapies have become an important treatment for a number of human malignancies. The VEGF inhibitors are actually effective in several types of cancers, however, the benefits are transiently, and the vast majority of patients who initially respond to the therapies will develop resistance. One of possible mechanisms for the acquired resistance may be the direct effect(s) of VEGF inhibitors on tumor cells expressing VEGF receptors (VEGFR). Thus, we investigated here the direct effect of chronic VEGF inhibition on phenotype changes in human colorectal cancer (CRC) cells. To chronically inhibit cancer cell-derived VEGF, human CRC cell lines (HCT116 and RKO) were chronically exposed (2 months) to an anti-VEGF monoclonal antibody (mAb) or were disrupted the Vegf gene (VEGF-KO). Effects of VEGF family members were blocked by treatment with a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI). Hypoxia-induced apoptosis under VEGF inhibited conditions was measured by TUNEL assay. Spheroid formation ability was assessed using a 3-D spheroid cell culture system. Chronic inhibition of secreted/extracellular VEGF by an anti-VEGF mAb redundantly increased VEGF family member (PlGF, VEGFR1 and VEGFR2), induced a resistance to hypoxia-induced apoptosis, and increased spheroid formation ability. This apoptotic resistance was partially abrogated by a VEGFR-TKI, which blocked the compensate pathway consisted of VEGF family members, or by knockdown of Vegf mRNA, which inhibited intracellular function(s) of all Vegf gene products. Interestingly, chronic and complete depletion of all Vegf gene products by Vegf gene knockout further augmented these phenotypes in the compensate pathway-independent manner. These accelerated phenotypes were significantly suppressed by knockdown of hypoxia-inducible factor-1α that was up-regulated in the VEGF-KO cell lines. Our findings suggest that chronic inhibition of tumor cell-derived VEGF

  7. MicroRNA and protein profiling of brain metastasis competent cell-derived exosomes.

    Directory of Open Access Journals (Sweden)

    Laura Camacho

    Full Text Available Exosomes are small membrane vesicles released by most cell types including tumor cells. The intercellular exchange of proteins and genetic material via exosomes is a potentially effective approach for cell-to-cell communication and it may perform multiple functions aiding to tumor survival and metastasis. We investigated microRNA and protein profiles of brain metastatic (BM versus non-brain metastatic (non-BM cell-derived exosomes. We studied the cargo of exosomes isolated from brain-tropic 70W, MDA-MB-231BR, and circulating tumor cell brain metastasis-selected markers (CTC1BMSM variants, and compared them with parental non-BM MeWo, MDA-MB-231P and CTC1P cells, respectively. By performing microRNA PCR array we identified one up-regulated (miR-210 and two down-regulated miRNAs (miR-19a and miR-29c in BM versus non-BM exosomes. Second, we analyzed the proteomic content of cells and exosomes isolated from these six cell lines, and detected high expression of proteins implicated in cell communication, cell cycle, and in key cancer invasion and metastasis pathways. Third, we show that BM cell-derived exosomes can be internalized by non-BM cells and that they effectively transport their cargo into cells, resulting in increased cell adhesive and invasive potencies. These results provide a strong rationale for additional investigations of exosomal proteins and miRNAs towards more profound understandings of exosome roles in brain metastasis biogenesis, and for the discovery and application of non-invasive biomarkers for new therapies combating brain metastasis.

  8. Induced pluripotent stem cell-derived cardiac progenitors differentiate to cardiomyocytes and form biosynthetic tissues.

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    Nicolas Christoforou

    Full Text Available The mammalian heart has little capacity to regenerate, and following injury the myocardium is replaced by non-contractile scar tissue. Consequently, increased wall stress and workload on the remaining myocardium leads to chamber dilation, dysfunction, and heart failure. Cell-based therapy with an autologous, epigenetically reprogrammed, and cardiac-committed progenitor cell source could potentially reverse this process by replacing the damaged myocardium with functional tissue. However, it is unclear whether cardiac progenitor cell-derived cardiomyocytes are capable of attaining levels of structural and functional maturity comparable to that of terminally-fated cardiomyocytes. Here, we first describe the derivation of mouse induced pluripotent stem (iPS cells, which once differentiated allow for the enrichment of Nkx2-5(+ cardiac progenitors, and the cardiomyocyte-specific expression of the red fluorescent protein. We show that the cardiac progenitors are multipotent and capable of differentiating into endothelial cells, smooth muscle cells and cardiomyocytes. Moreover, cardiac progenitor selection corresponds to cKit(+ cell enrichment, while cardiomyocyte cell-lineage commitment is concomitant with dual expression of either cKit/Flk1 or cKit/Sca-1. We proceed to show that the cardiac progenitor-derived cardiomyocytes are capable of forming electrically and mechanically coupled large-scale 2D cell cultures with mature electrophysiological properties. Finally, we examine the cell progenitors' ability to form electromechanically coherent macroscopic tissues, using a physiologically relevant 3D culture model and demonstrate that following long-term culture the cardiomyocytes align, and form robust electromechanical connections throughout the volume of the biosynthetic tissue construct. We conclude that the iPS cell-derived cardiac progenitors are a robust cell source for tissue engineering applications and a 3D culture platform for pharmacological

  9. Improved flow cytometric assessment reveals distinct microvesicle (cell-derived microparticle signatures in joint diseases.

    Directory of Open Access Journals (Sweden)

    Bence György

    Full Text Available INTRODUCTION: Microvesicles (MVs, earlier referred to as microparticles, represent a major type of extracellular vesicles currently considered as novel biomarkers in various clinical settings such as autoimmune disorders. However, the analysis of MVs in body fluids has not been fully standardized yet, and there are numerous pitfalls that hinder the correct assessment of these structures. METHODS: In this study, we analyzed synovial fluid (SF samples of patients with osteoarthritis (OA, rheumatoid arthritis (RA and juvenile idiopathic arthritis (JIA. To assess factors that may confound MV detection in joint diseases, we used electron microscopy (EM, Nanoparticle Tracking Analysis (NTA and mass spectrometry (MS. For flow cytometry, a method commonly used for phenotyping and enumeration of MVs, we combined recent advances in the field, and used a novel approach of differential detergent lysis for the exclusion of MV-mimicking non-vesicular signals. RESULTS: EM and NTA showed that substantial amounts of particles other than MVs were present in SF samples. Beyond known MV-associated proteins, MS analysis also revealed abundant plasma- and immune complex-related proteins in MV preparations. Applying improved flow cytometric analysis, we demonstrate for the first time that CD3(+ and CD8(+ T-cell derived SF MVs are highly elevated in patients with RA compared to OA patients (p=0.027 and p=0.009, respectively, after Bonferroni corrections. In JIA, we identified reduced numbers of B cell-derived MVs (p=0.009, after Bonferroni correction. CONCLUSIONS: Our results suggest that improved flow cytometric assessment of MVs facilitates the detection of previously unrecognized disease-associated vesicular signatures.

  10. The differentiation of embryonic stem cells seeded on electrospun nanofibers into neural lineages.

    Science.gov (United States)

    Xie, Jingwei; Willerth, Stephanie M; Li, Xiaoran; Macewan, Matthew R; Rader, Allison; Sakiyama-Elbert, Shelly E; Xia, Younan

    2009-01-01

    Due to advances in stem cell biology, embryonic stem (ES) cells can be induced to differentiate into a particular mature cell lineage when cultured as embryoid bodies. Although transplantation of ES cells-derived neural progenitor cells has been demonstrated with some success for either spinal cord injury repair in small animal model, control of ES cell differentiation into complex, viable, higher ordered tissues is still challenging. Mouse ES cells have been induced to become neural progenitors by adding retinoic acid to embryoid body cultures for 4 days. In this study, we examine the use of electrospun biodegradable polymers as scaffolds not only for enhancing the differentiation of mouse ES cells into neural lineages but also for promoting and guiding the neurite outgrowth. A combination of electrospun fiber scaffolds and ES cells-derived neural progenitor cells could lead to the development of a better strategy for nerve injury repair.

  11. A systemized approach to investigate Ca2+ synchronization in clusters of human induced pluripotent stem-cell derived cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Aled R Jones

    2016-01-01

    Full Text Available Induced pluripotent stem cell-derived cardiomyocytes (IPS-CM are considered by many to be the cornerstone of future approaches to repair the diseased heart. However, current methods for producing IPS-CM typically yield highly variable populations with low batch-to-batch reproducibility. The underlying reasons for this are not fully understood. Here we report on a systematized approach to investigate the effect of maturation in embryoid bodies (EB versus ‘on plate’ culture on spontaneous activity and regional Ca2+ synchronization in IPS-CM clusters. A detailed analysis of the temporal and spatial organization of Ca2+ spikes in IPS-CM clusters revealed that the disaggregation of EBs between 0.5 and 2 weeks produced IPS-CM characterized by spontaneous beating and high levels of regional Ca2+ synchronization. These phenomena were typically absent in IPS-CM obtained from older EBs (> 2 weeks. The maintenance of all spontaneously active IPS-CM clusters under ‘on plate’ culture conditions promoted the progressive reduction in regional Ca2+ synchronization and the loss of spontaneous Ca2+ spiking. Raising the extracellular [Ca2+] surrounding these quiescent IPS-CM clusters from approximately 0.4 to 1.8 mM unmasked discrete behaviours typified by either a long-lasting Ca2+ elevation that returned to baseline or b persistent, large-amplitude Ca2+ oscillations around an increased cytoplasmic [Ca2+]. The different responses of IPS-CM to elevated extracellular [Ca2+] could be traced back to their routes of derivation. The data point to the possibility of predictably influencing IPS-CM phenotype and response to external activation via defined interventions at early stages in their maturation.

  12. Characterization of single cell derived cultures of periosteal progenitor cells to ensure the cell quality for clinical application.

    Directory of Open Access Journals (Sweden)

    Stefan Stich

    Full Text Available For clinical applications of cells and tissue engineering products it is of importance to characterize the quality of the used cells in detail. Progenitor cells from the periosteum are already routinely applied in the clinics for the regeneration of the maxillary bone. Periosteal cells have, in addition to their potential to differentiate into bone, the ability to develop into cartilage and fat. However, the question arises whether all cells isolated from periosteal biopsies are able to differentiate into all three tissue types, or whether there are subpopulations. For an efficient and approved application in bone or cartilage regeneration the clarification of this question is of interest. Therefore, 83 different clonal cultures of freshly isolated human periosteal cells derived from mastoid periosteum biopsies of 4 donors were generated and growth rates calculated. Differentiation capacities of 51 clonal cultures towards the osteogenic, the chondrogenic, and the adipogenic lineage were investigated. Histological and immunochemical stainings showed that 100% of the clonal cultures differentiated towards the osteogenic lineage, while 94.1% demonstrated chondrogenesis, and 52.9% could be stimulated to adipogenesis. For osteogenesis real-time polymerase chain reaction (PCR of BGLAP and RUNX2 and for adipogenesis of FABP4 and PPARG confirmed the results. Overall, 49% of the cells exhibited a tripotent potential, 45.1% showed a bipotent potential (without adipogenic differentiation, 3.9% bipotent (without chondrogenic differentiation, and 2% possessed a unipotent osteogenic potential. In FACS analyses, no differences in the marker profile of undifferentiated clonal cultures with bi- and tripotent differentiation capacity were found. Genome-wide microarray analysis revealed 52 differentially expressed genes for clonal subpopulations with or without chondrogenic differentiation capacity, among them DCN, NEDD9, TGFBR3, and TSLP. For clinical

  13. Identification and purification of human induced pluripotent stem cell-derived atrial-like cardiomyocytes based on sarcolipin expression.

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    Rebecca Josowitz

    Full Text Available The use of human stem cell-derived cardiomyocytes to study atrial biology and disease has been restricted by the lack of a reliable method for stem cell-derived atrial cell labeling and purification. The goal of this study was to generate an atrial-specific reporter construct to identify and purify human stem cell-derived atrial-like cardiomyocytes. We have created a bacterial artificial chromosome (BAC reporter construct in which fluorescence is driven by expression of the atrial-specific gene sarcolipin (SLN. When purified using flow cytometry, cells with high fluorescence specifically express atrial genes and display functional calcium handling and electrophysiological properties consistent with atrial cardiomyocytes. Our data indicate that SLN can be used as a marker to successfully monitor and isolate hiPSC-derived atrial-like cardiomyocytes. These purified cells may find many applications, including in the study of atrial-specific pathologies and chamber-specific lineage development.

  14. CD133+ cells derived from skeletal muscles of Duchenne muscular dystrophy patients have a compromised myogenic and muscle regenerative capability.

    Science.gov (United States)

    Meng, Jinhong; Muntoni, Francesco; Morgan, Jennifer

    2018-05-12

    Cell-mediated gene therapy is a possible means to treat muscular dystrophies like Duchenne muscular dystrophy. Autologous patient stem cells can be genetically-corrected and transplanted back into the patient, without causing immunorejection problems. Regenerated muscle fibres derived from these cells will express the missing dystrophin protein, thus improving muscle function. CD133+ cells derived from normal human skeletal muscle contribute to regenerated muscle fibres and form muscle stem cells after their intra-muscular transplantation into an immunodeficient mouse model. But it is not known whether CD133+ cells derived from DMD patient muscles have compromised muscle regenerative function. To test this, we compared CD133+ cells derived from DMD and normal human muscles. DMD CD133+ cells had a reduced capacity to undergo myogenic differentiation in vitro compared with CD133+ cells derived from normal muscle. In contrast to CD133+ cells derived from normal human muscle, those derived from DMD muscle formed no satellite cells and gave rise to significantly fewer muscle fibres of donor origin, after their intra-muscular transplantation into an immunodeficient, non-dystrophic, mouse muscle. DMD CD133+ cells gave rise to more clones of smaller size and more clones that were less myogenic than did CD133+ cells derived from normal muscle. The heterogeneity of the progeny of CD133+ cells, combined with the reduced proliferation and myogenicity of DMD compared to normal CD133+ cells, may explain the reduced regenerative capacity of DMD CD133+ cells. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

  15. FZD4 Marks Lateral Plate Mesoderm and Signals with NORRIN to Increase Cardiomyocyte Induction from Pluripotent Stem Cell-Derived Cardiac Progenitors

    Directory of Open Access Journals (Sweden)

    Charles Yoon

    2018-01-01

    Full Text Available The identification of cell surface proteins on stem cells or stem cell derivatives is a key strategy for the functional characterization, isolation, and understanding of stem cell population dynamics. Here, using an integrated mass spectrometry- and microarray-based approach, we analyzed the surface proteome and transcriptome of cardiac progenitor cells (CPCs generated from the stage-specific differentiation of mouse and human pluripotent stem cells. Through bioinformatics analysis, we have identified and characterized FZD4 as a marker for lateral plate mesoderm. Additionally, we utilized FZD4, in conjunction with FLK1 and PDGFRA, to further purify CPCs and increase cardiomyocyte (CM enrichment in both mouse and human systems. Moreover, we have shown that NORRIN presented to FZD4 further increases CM output via proliferation through the canonical WNT pathway. Taken together, these findings demonstrate a role for FZD4 in mammalian cardiac development.

  16. Study of mesanchymal stem cells derived from human umbilical cord vein wall and determining the Process of differentiation to cartilage and bone

    Directory of Open Access Journals (Sweden)

    MohammadAli Zare

    2015-01-01

    Full Text Available Background: Mesenchymal stem cells (MSCs comprise a rare population of multipotent progenitors capable of supporting hematopoiesis and differentiating into three (osteogenic, adipogenic, and chondrogenic or more (myogenic, cardiomyogenic, etc. lineages. Due to this ability, MSCs appear to be an attractive tool in the context of tissue engineering and cell-based therapy. Currently, bone marrow represents the main source of MSCs for both experimental and clinical studies. The purpose of this study was isolation and quantitative comparison of mesenchymal stem cells derived from umbilical vein. Materials and Methods: In this study, 35 samples of umbilical cord of healthy full- term newborn were studied. Results: The cells had fibroblastoid like appearance and had revealed the potential to differentiate into three linage of bone, Adipose and cartilage. Surface markers for mesenchymal nature were their demonstratives. Conclusion: Based on our findings the mesenchymal stem cells, from umbilical vein wall can be isolated, cultured and differentiated into three categories of bone, cartilage and adipose.

  17. Levels of PAH-DNA adducts in cord blood and cord tissue and the risk of fetal neural tube defects in a Chinese population.

    Science.gov (United States)

    Yi, Deqing; Yuan, Yue; Jin, Lei; Zhou, Guodong; Zhu, Huiping; Finnell, Richard H; Ren, Aiguo

    2015-01-01

    Maternal exposure to polycyclic aromatic hydrocarbons (PAHs) has been shown to be associated with an elevated risk for neural tube defects (NTDs). In the human body, PAHs are bioactivated and the resultant reactive epoxides can covalently bind to DNA to form PAH-DNA adducts, which may, in turn, cause transcription errors, changes in gene expression or altered patterns of apoptosis. During critical developmental phases, these changes can result in abnormal morphogenesis. We aimed to examine the relationship between the levels of PAH-DNA adducts in cord blood and cord tissue and the risk of NTDs. From 2010 to 2012, 60 NTD cases and 60 healthy controls were recruited from a population-based birth defects surveillance system in five counties of Shanxi Province in Northern China, where the emission of PAHs remains one of the highest in the country and PAHs exposure is highly prevalent. PAH-DNA adducts in cord blood of 15 NTD cases and 15 control infants, and in cord tissue of 60 NTD cases and 60 control infants were measured using the (32)P-postlabeling method. PAH-DNA adduct levels in cord blood tend to be higher in the NTD group (28.5 per 10(8) nucleotides) compared with controls (19.7 per 10(8) nucleotides), although the difference was not statistically significant (P=0.377). PAH-DNA adducts in cord tissue were significantly higher in the NTD group (24.6 per 10(6) nucleotides) than in the control group (15.3 per 10(6) nucleotides), P=0.010. A positive dose-response relationship was found between levels of PAH-DNA adducts in cord tissue and the risk of NTDs (P=0.009). When the lowest tertile was used as the referent and potential confounding factors were adjusted for, a 1.03-fold (95% CI, 0.37-2.89) and 2.96-fold (95% CI, 1.16-7.58) increase in the risk of NTDs was observed for fetuses whose cord tissue PAH-DNA adduct levels were in the second and highest tertile, respectively. High levels of PAH-DNA adducts in fetal tissues were associated with increased risks of

  18. Use of human stem cell derived cardiomyocytes to examine sunitinib mediated cardiotoxicity and electrophysiological alterations

    Energy Technology Data Exchange (ETDEWEB)

    Cohen, J.D., E-mail: jennifer.cohen@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States); Babiarz, J.E., E-mail: joshua.babiarz@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States); Abrams, R.M., E-mail: rory.abrams@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States); Guo, L., E-mail: liang.guo@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States); Kameoka, S., E-mail: sei.kameoka@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States); Chiao, E., E-mail: eric.chiao@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States); Taunton, J., E-mail: taunton@cmp.ucsf.edu [Howard Hughes Medical Institute, Cellular and Molecular Pharmacology, University California San Francisco, San Francisco, CA 94158 (United States); Kolaja, K.L., E-mail: kyle.kolaja@roche.com [Early and Investigative Safety, Nonclinical Safety, Hoffmann-La Roche, 340 Kingsland Street, Nutley, NJ 07110 (United States)

    2011-11-15

    Sunitinib, an oral tyrosine kinase inhibitor approved to treat advanced renal cell carcinoma and gastrointestinal stroma tumor, is associated with clinical cardiac toxicity. Although the precise mechanism of sunitinib cardiotoxicity is not known, both the key metabolic energy regulator, AMP-activated protein kinase (AMPK), and ribosomal S 6 kinase (RSK) have been hypothesized as causative, albeit based on rodent models. To study the mechanism of sunitinib-mediated cardiotoxicity in a human model, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) having electrophysiological and contractile properties of native cardiac tissue were investigated. Sunitinib was cardiotoxic in a dose-dependent manner with an IC{sub 50} in the low micromolar range, observed by a loss of cellular ATP, an increase in oxidized glutathione, and induction of apoptosis in iPSC-CMs. Pretreatment of iPSC-CMs with AMPK activators AICAR or metformin, increased the phosphorylation of pAMPK-T172 and pACC-S79, but only marginally attenuated sunitinib mediated cell death. Furthermore, additional inhibitors of AMPK were not directly cytotoxic to iPSC-CMs up to 250 {mu}M concentrations. Inhibition of RSK with a highly specific, irreversible, small molecule inhibitor (RSK-FMK-MEA) did not induce cytotoxicity in iPSC-CMs below 250 {mu}M. Extensive electrophysiological analysis of sunitinib and RSK-FMK-MEA mediated conduction effects were performed. Taken together, these findings suggest that inhibition of AMPK and RSK are not a major component of sunitinib-induced cardiotoxicity. Although the exact mechanism of cardiotoxicity of sunitinib is not known, it is likely due to inhibition of multiple kinases simultaneously. These data highlight the utility of human iPSC-CMs in investigating the potential molecular mechanisms underlying drug-induced cardiotoxicity. -- Highlights: Black-Right-Pointing-Pointer Cytoxic effect of sunitinib on human stem cell derived cardiomyocytes Black

  19. Use of human stem cell derived cardiomyocytes to examine sunitinib mediated cardiotoxicity and electrophysiological alterations

    International Nuclear Information System (INIS)

    Cohen, J.D.; Babiarz, J.E.; Abrams, R.M.; Guo, L.; Kameoka, S.; Chiao, E.; Taunton, J.; Kolaja, K.L.

    2011-01-01

    Sunitinib, an oral tyrosine kinase inhibitor approved to treat advanced renal cell carcinoma and gastrointestinal stroma tumor, is associated with clinical cardiac toxicity. Although the precise mechanism of sunitinib cardiotoxicity is not known, both the key metabolic energy regulator, AMP-activated protein kinase (AMPK), and ribosomal S 6 kinase (RSK) have been hypothesized as causative, albeit based on rodent models. To study the mechanism of sunitinib-mediated cardiotoxicity in a human model, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) having electrophysiological and contractile properties of native cardiac tissue were investigated. Sunitinib was cardiotoxic in a dose-dependent manner with an IC 50 in the low micromolar range, observed by a loss of cellular ATP, an increase in oxidized glutathione, and induction of apoptosis in iPSC-CMs. Pretreatment of iPSC-CMs with AMPK activators AICAR or metformin, increased the phosphorylation of pAMPK-T172 and pACC-S79, but only marginally attenuated sunitinib mediated cell death. Furthermore, additional inhibitors of AMPK were not directly cytotoxic to iPSC-CMs up to 250 μM concentrations. Inhibition of RSK with a highly specific, irreversible, small molecule inhibitor (RSK-FMK-MEA) did not induce cytotoxicity in iPSC-CMs below 250 μM. Extensive electrophysiological analysis of sunitinib and RSK-FMK-MEA mediated conduction effects were performed. Taken together, these findings suggest that inhibition of AMPK and RSK are not a major component of sunitinib-induced cardiotoxicity. Although the exact mechanism of cardiotoxicity of sunitinib is not known, it is likely due to inhibition of multiple kinases simultaneously. These data highlight the utility of human iPSC-CMs in investigating the potential molecular mechanisms underlying drug-induced cardiotoxicity. -- Highlights: ► Cytoxic effect of sunitinib on human stem cell derived cardiomyocytes ► Sunitinib causes ATP depletion, LDH release, GSH

  20. Evaluation of mRNA expression levels and electrophysiological function of neuron-like cells derived from canine bone marrow stromal cells.

    Science.gov (United States)

    Nakano, Rei; Edamura, Kazuya; Sugiya, Hiroshi; Narita, Takanori; Okabayashi, Ken; Moritomo, Tadaaki; Teshima, Kenji; Asano, Kazushi; Nakayama, Tomohiro

    2013-10-01

    To investigate the in vitro differentiation of canine bone marrow stromal cells (BMSCs) into functional, mature neurons. Bone marrow from 6 adult dogs. BMSCs were isolated from bone marrow and chemically induced to develop into neurons. The morphology of the BMSCs during neuronal induction was monitored, and immunocytochemical analyses for neuron markers were performed after the induction. Real-time PCR methods were used to evaluate the mRNA expression levels of markers for neural stem or progenitor cells, neurons, and ion channels, and western blotting was used to assess the expression of neuronal proteins before and after neuronal induction. The electrophysiological properties of the neuron-like cells induced from canine BMSCs were evaluated with fluorescent dye to monitor Ca(2)+ influx. Canine BMSCs developed a neuron-like morphology after neuronal induction. Immunocytochemical analysis revealed that these neuron-like cells were positive for neuron markers. After induction, the cells' mRNA expression levels of almost all neuron and ion channel markers increased, and the protein expression levels of nestin and neurofilament-L increased significantly. However, the neuron-like cells derived from canine BMSCs did not have the Ca(2)+ influx characteristic of spiking neurons. Although canine BMSCs had neuron-like morphological and biochemical properties after induction, they did not develop the electrophysiological characteristics of neurons. Thus, these results have suggested that canine BMSCs could have the capacity to differentiate into a neuronal lineage, but the differentiation protocol used may have been insufficient to induce development into functional neurons.

  1. Puerarin Facilitates T-Tubule Development of Murine Embryonic Stem Cell-Derived Cardiomyocytes

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    Lu Wang

    2014-07-01

    Full Text Available Aims: The embryonic stem cell-derived cardiomyocytes (ES-CM is one of the promising cell sources for repopulation of damaged myocardium. However, ES-CMs present immature structure, which impairs their integration with host tissue and functional regeneration. This study used murine ES-CMs as an in vitro model of cardiomyogenesis to elucidate the effect of puerarin, the main compound found in the traditional Chinese medicine the herb Radix puerariae, on t-tubule development of murine ES-CMs. Methods: Electron microscope was employed to examine the ultrastructure. The investigation of transverse-tubules (t-tubules was performed by Di-8-ANEPPS staining. Quantitative real-time PCR was utilized to study the transcript level of genes related to t-tubule development. Results: We found that long-term application of puerarin throughout cardiac differentiation improved myofibril array and sarcomeres formation, and significantly facilitated t-tubules development of ES-CMs. The transcript levels of caveolin-3, amphiphysin-2 and junctophinlin-2, which are crucial for the formation and development of t-tubules, were significantly upregulated by puerarin treatment. Furthermore, puerarin repressed the expression of miR-22, which targets to caveolin-3. Conclusion: Our data showed that puerarin facilitates t-tubule development of murine ES-CMs. This might be related to the repression of miR-22 by puerarin and upregulation of Cav3, Bin1 and JP2 transcripts.

  2. Efficient Generation of Human Embryonic Stem Cell-Derived Corneal Endothelial Cells by Directed Differentiation.

    Directory of Open Access Journals (Sweden)

    Kathryn L McCabe

    Full Text Available To generate human embryonic stem cell derived corneal endothelial cells (hESC-CECs for transplantation in patients with corneal endothelial dystrophies.Feeder-free hESC-CECs were generated by a directed differentiation protocol. hESC-CECs were characterized by morphology, expression of corneal endothelial markers, and microarray analysis of gene expression.hESC-CECs were nearly identical morphologically to primary human corneal endothelial cells, expressed Zona Occludens 1 (ZO-1 and Na+/K+ATPaseα1 (ATPA1 on the apical surface in monolayer culture, and produced the key proteins of Descemet's membrane, Collagen VIIIα1 and VIIIα2 (COL8A1 and 8A2. Quantitative PCR analysis revealed expression of all corneal endothelial pump transcripts. hESC-CECs were 96% similar to primary human adult CECs by microarray analysis.hESC-CECs are morphologically similar, express corneal endothelial cell markers and express a nearly identical complement of genes compared to human adult corneal endothelial cells. hESC-CECs may be a suitable alternative to donor-derived corneal endothelium.

  3. Functional Studies of Missense TREM2 Mutations in Human Stem Cell-Derived Microglia

    Directory of Open Access Journals (Sweden)

    Philip W. Brownjohn

    2018-04-01

    Full Text Available Summary: The derivation of microglia from human stem cells provides systems for understanding microglial biology and enables functional studies of disease-causing mutations. We describe a robust method for the derivation of human microglia from stem cells, which are phenotypically and functionally comparable with primary microglia. We used stem cell-derived microglia to study the consequences of missense mutations in the microglial-expressed protein triggering receptor expressed on myeloid cells 2 (TREM2, which are causal for frontotemporal dementia-like syndrome and Nasu-Hakola disease. We find that mutant TREM2 accumulates in its immature form, does not undergo typical proteolysis, and is not trafficked to the plasma membrane. However, in the absence of plasma membrane TREM2, microglia differentiate normally, respond to stimulation with lipopolysaccharide, and are phagocytically competent. These data indicate that dementia-associated TREM2 mutations have subtle effects on microglia biology, consistent with the adult onset of disease in individuals with these mutations. : Brownjohn and colleagues report methods to generate microglia from induced pluripotent human stem cells, which they demonstrate are highly similar to cultured primary human microglia. Microglia differentiated from patient-derived stem cells carrying neurological disease-causing mutations in the TREM2 receptor differentiate normally and respond appropriately to pathogenic stimuli, despite the absence of functional TREM2 receptor on the plasma membrane. Keywords: dementia, microglia, TREM2, Nasu-Hakola disease, frontotemporal dementia, iPSC-microglia, neuroinflammation

  4. Analysis of mammary specific gene locus regulation in differentiated cells derived by somatic cell fusion

    International Nuclear Information System (INIS)

    Robinson, Claire; Kolb, Andreas F.

    2009-01-01

    The transcriptional regulation of a gene is best analysed in the context of its normal chromatin surroundings. However, most somatic cells, in contrast to embryonic stem cells, are refractory to accurate modification by homologous recombination. We show here that it is possible to introduce precise genomic modifications in ES cells and to analyse the phenotypic consequences in differentiated cells by using a combination of gene targeting, site-specific recombination and somatic cell fusion. To provide a proof of principle, we have analysed the regulation of the casein gene locus in mammary gland cells derived from modified murine ES cells by somatic cell fusion. A β-galactosidase reporter gene was inserted in place of the β-casein gene and the modified ES cells, which do not express the reporter gene, were fused with the mouse mammary gland cell line HC11. The resulting cell clones expressed the β-galactosidase gene to a similar extent and with similar hormone responsiveness as the endogenous gene. However, a reporter gene under the control of a minimal β-casein promoter (encompassing the two consensus STAT5 binding sites which mediate the hormone response of the casein genes) was unable to replicate expression levels or hormone responsiveness of the endogenous gene when inserted into the same site of the casein locus. As expected, these results implicate sequences other than the STAT5 sites in the regulation of the β-casein gene

  5. Mesenchymal Stem Cell-Derived Factors Restore Function to Human Frataxin-Deficient Cells.

    Science.gov (United States)

    Kemp, Kevin; Dey, Rimi; Cook, Amelia; Scolding, Neil; Wilkins, Alastair

    2017-08-01

    Friedreich's ataxia is an inherited neurological disorder characterised by mitochondrial dysfunction and increased susceptibility to oxidative stress. At present, no therapy has been shown to reduce disease progression. Strategies being trialled to treat Friedreich's ataxia include drugs that improve mitochondrial function and reduce oxidative injury. In addition, stem cells have been investigated as a potential therapeutic approach. We have used siRNA-induced knockdown of frataxin in SH-SY5Y cells as an in vitro cellular model for Friedreich's ataxia. Knockdown of frataxin protein expression to levels detected in patients with the disorder was achieved, leading to decreased cellular viability, increased susceptibility to hydrogen peroxide-induced oxidative stress, dysregulation of key anti-oxidant molecules and deficiencies in both cell proliferation and differentiation. Bone marrow stem cells are being investigated extensively as potential treatments for a wide range of neurological disorders, including Friedreich's ataxia. The potential neuroprotective effects of bone marrow-derived mesenchymal stem cells were therefore studied using our frataxin-deficient cell model. Soluble factors secreted by mesenchymal stem cells protected against cellular changes induced by frataxin deficiency, leading to restoration in frataxin levels and anti-oxidant defences, improved survival against oxidative stress and stimulated both cell proliferation and differentiation down the Schwann cell lineage. The demonstration that mesenchymal stem cell-derived factors can restore cellular homeostasis and function to frataxin-deficient cells further suggests that they may have potential therapeutic benefits for patients with Friedreich's ataxia.

  6. Three-dimensional bioprinting of stem-cell derived tissues for human regenerative medicine.

    Science.gov (United States)

    Skeldon, Gregor; Lucendo-Villarin, Baltasar; Shu, Wenmiao

    2018-07-05

    Stem cell technology in regenerative medicine has the potential to provide an unlimited supply of cells for drug testing, medical transplantation and academic research. In order to engineer a realistic tissue model using stem cells as an alternative to human tissue, it is essential to create artificial stem cell microenvironment or niches. Three-dimensional (3D) bioprinting is a promising tissue engineering field that offers new opportunities to precisely place stem cells within their niches layer-by-layer. This review covers bioprinting technologies, the current development of 'bio-inks' and how bioprinting has already been applied to stem-cell culture, as well as their applications for human regenerative medicine. The key considerations for bioink properties such as stiffness, stability and biodegradation, biocompatibility and printability are highlighted. Bioprinting of both adult and pluriopotent stem cells for various types of artificial tissues from liver to brain has been reviewed. 3D bioprinting of stem-cell derived tissues for human regenerative medicine is an exciting emerging area that represents opportunities for new research, industries and products as well as future challenges in clinical translation.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'. © 2018 The Author(s).

  7. Steps toward Maturation of Embryonic Stem Cell-Derived Cardiomyocytes by Defined Physical Signals

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    Nian Shen

    2017-07-01

    Full Text Available Cardiovascular disease remains a leading cause of mortality and morbidity worldwide. Embryonic stem cell-derived cardiomyocytes (ESC-CMs may offer significant advances in creating in vitro cardiac tissues for disease modeling, drug testing, and elucidating developmental processes; however, the induction of ESCs to a more adult-like CM phenotype remains challenging. In this study, we developed a bioreactor system to employ pulsatile flow (1.48 mL/min, cyclic strain (5%, and extended culture time to improve the maturation of murine and human ESC-CMs. Dynamically-cultured ESC-CMs showed an increased expression of cardiac-associated proteins and genes, cardiac ion channel genes, as well as increased SERCA activity and a Raman fingerprint with the presence of maturation-associated peaks similar to primary CMs. We present a bioreactor platform that can serve as a foundation for the development of human-based cardiac in vitro models to verify drug candidates, and facilitates the study of cardiovascular development and disease.

  8. Low antigenicity of hematopoietic progenitor cells derived from human ES cells

    Directory of Open Access Journals (Sweden)

    Eun-Mi Kim

    2010-02-01

    Full Text Available Eun-Mi Kim1, Nicholas Zavazava1,21Department of Internal Medicine, University of Iowa and Veterans Affairs Medical Center, Iowa City, Iowa, USA; 2Immunology Graduate Program, University of Iowa, Iowa City, Iowa, USAAbstract: Human embryonic stem (hES cells are essential for improved understanding of diseases and our ability to probe new therapies for use in humans. Currently, bone marrow cells and cord blood cells are used for transplantation into patients with hematopoietic malignancies, immunodeficiencies and in some cases for the treatment of autoimmune diseases. However, due to the high immunogenicity of these hematopoietic cells, toxic regimens of drugs are required for preconditioning and prevention of rejection. Here, we investigated the efficiency of deriving hematopoietic progenitor cells (HPCs from the hES cell line H13, after co-culturing with the murine stromal cell line OP9. We show that HPCs derived from the H13 ES cells poorly express major histocompatibility complex (MHC class I and no detectable class II antigens (HLA-DR. These characteristics make hES cell-derived hematopoietic cells (HPCs ideal candidates for transplantation across MHC barriers under minimal immunosuppression.Keywords: human embryonic stem cells, H13, hematopoiesis, OP9 stromal cells, immunogenicity

  9. Autologous Pluripotent Stem Cell-Derived β-Like Cells for Diabetes Cellular Therapy.

    Science.gov (United States)

    Millman, Jeffrey R; Pagliuca, Felicia W

    2017-05-01

    Development of stem cell technologies for cell replacement therapy has progressed rapidly in recent years. Diabetes has long been seen as one of the first applications for stem cell-derived cells because of the loss of only a single cell type-the insulin-producing β-cell. Recent reports have detailed strategies that overcome prior hurdles to generate functional β-like cells from human pluripotent stem cells in vitro, including from human induced pluripotent stem cells (hiPSCs). Even with this accomplishment, addressing immunological barriers to transplantation remains a major challenge for the field. The development of clinically relevant hiPSC derivation methods from patients and demonstration that these cells can be differentiated into β-like cells presents a new opportunity to treat diabetes without immunosuppression or immunoprotective encapsulation or with only targeted protection from autoimmunity. This review focuses on the current status in generating and transplanting autologous β-cells for diabetes cell therapy, highlighting the unique advantages and challenges of this approach. © 2017 by the American Diabetes Association.

  10. Appearance of differentiated cells derived from polar body nuclei in the silkworm, Bombyx mori

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    Hiroki eSakai

    2013-09-01

    Full Text Available AbstractIn Bombyx mori, polar body nuclei are observed until 9h after egg lying, however, the fate of polar body nuclei remains unclear. To examine the fate of polar body nuclei, we employed a mutation of serosal cell pigmentation, pink-eyed white egg (pe. The heterozygous pe/+pe females produced black serosal cells in white eggs, while pe/pe females did not produce black serosal cells in white eggs. These results suggest that the appearance of black serosal cells in white eggs depends on the genotype (pe/ +pe of the mother. Because the polar body nuclei had +pe genes in the white eggs laid by a pe/ +pe female, polar body nuclei participate in development and differentiate into functional cell (serosal cells. Analyses of serosal cells pigmentation indicated that approximately 30% of the eggs contained polar-body-nucleus-derived cells. These results demonstrate that polar-body-nucleus-derived cells appeared at a high frequency under natural conditions. Approximately 80% of polar-body-nucleus-derived cells appeared near the anterior pole and the dorsal side, which is opposite to where embryogenesis occurs. The number of cells derived from the polar body nuclei was very low. Approximately 26 % of these eggs contained only one black serosal cell. PCR-based analysis revealed that the polar-body-nucleus-derived cells disappeared in late embryonic stages (stage 25. Overall, polar-body-nuclei-derived cells were unlikely to contribute to embryos.

  11. Engineered Microenvironments for the Maturation and Observation of Human Embryonic Stem Cell Derived Cardiomyocytes

    Science.gov (United States)

    Salick, Max R.

    The human heart is a dynamic system that undergoes substantial changes as it develops and adapts to the body's growing needs. To better understand the physiology of the heart, researchers have begun to produce immature heart muscle cells, or cardiomyocytes, from pluripotent stem cell sources with remarkable efficiency. These stem cell-derived cardiomyocytes hold great potential in the understanding and treatment of heart disease; however, even after prolonged culture, these cells continue to exhibit an immature phenotype, as indicated by poor sarcomere organization and calcium handling, among other features. The lack of maturation that is observed in these cardiomyocytes greatly limits their applicability towards drug screening, disease modeling, and cell therapy applications. The mechanical environment surrounding a cell has been repeatedly shown to have a large impact on that cell's behavior. For this reason, we have implemented micropatterning methods to mimic the level of alignment that occurs in the heart in vivo in order to study how this alignment may help the cells to produce a more mature sarcomere phenotype. It was discovered that the level of sarcomere organization of a cardiomyocyte can be strongly influenced by the micropattern lane geometry on which it adheres. Steps were taken to optimize this micropattern platform, and studies of protein organization, gene expression, and myofibrillogenesis were conducted. Additionally, a set of programs was developed to provide quantitative analysis of the level of sarcomere organization, as well as to assist with several other tissue engineering applications.

  12. Induced Pluripotent Stem Cell-Derived Red Blood Cells and Platelet Concentrates: From Bench to Bedside.

    Science.gov (United States)

    Focosi, Daniele; Amabile, Giovanni

    2017-12-27

    Red blood cells and platelets are anucleate blood components indispensable for oxygen delivery and hemostasis, respectively. Derivation of these blood elements from induced pluripotent stem (iPS) cells has the potential to develop blood donor-independent and genetic manipulation-prone products to complement or replace current transfusion banking, also minimizing the risk of alloimmunization. While the production of erythrocytes from iPS cells has challenges to overcome, such as differentiation into adult-type phenotype that functions properly after transfusion, platelet products are qualitatively and quantitatively approaching a clinically-applicable level owing to advances in expandable megakaryocyte (MK) lines, platelet-producing bioreactors, and novel reagents. Guidelines that assure the quality of iPS cells-derived blood products for clinical application represent a novel challenge for regulatory agencies. Considering the minimal risk of tumorigenicity and the expected significant demand of such products, ex vivo production of iPS-derived blood components can pave the way for iPS translation into the clinic.

  13. Effect of strenuous physical exercise on circulating cell-derived microparticles.

    Science.gov (United States)

    Chaar, Vicky; Romana, Marc; Tripette, Julien; Broquere, Cédric; Huisse, Marie-Geneviève; Hue, Olivier; Hardy-Dessources, Marie-Dominique; Connes, Philippe

    2011-01-01

    Strenuous exercise is associated with an inflammatory response involving the activation of several types of blood cells. In order to document the specific activation of these cell types, we studied the effect of three maximal exercise tests conducted to exhaustion on the quantitative and qualitative pattern of circulating cell-derived microparticles and inflammatory molecules in healthy subjects. This study mainly indicated that the plasma concentration of microparticles from platelets and polymorphonuclear neutrophils (PMN) was increased immediately after the strenuous exercise. In addition, the increase in plasma concentration of microparticles from PMN and platelets was still observed after 2 hours of recovery. A similar pattern was observed for the IL-6 plasma level. In contrast, no change was observed for either soluble selectins or plasma concentration of microparticles from red blood cells, monocytes and endothelial cells. In agreement, sVCAM-1 and sICAM-1 levels were not changed by the exercise. We conclude that a strenuous exercise is accompanied by platelet- and PMN-derived microparticle production that probably reflects the activation of these two cell types.

  14. A simple clot based assay for detection of procoagulant cell-derived microparticles.

    Science.gov (United States)

    Patil, Rucha; Ghosh, Kanjaksha; Shetty, Shrimati

    2016-05-01

    Cell-derived microparticles (MPs) are important biomarkers in many facets of medicine. However, the MP detection methods used till date are costly and time consuming. The main aim of this study was to standardize an in-house clot based screening method for MP detection which would not only be specific and sensitive, but also inexpensive. Four different methods of MP assessment were performed and the results correlated. Using the flow cytometry technique as the gold standard, 25 samples with normal phosphatidylserine (PS) expressing MP levels and 25 samples with elevated levels were selected, which was cross checked by the commercial STA Procoag PPL clotting time (CT) assay. A simple recalcification time and an in-house clot assay were the remaining two tests. The in-house test measures the CT after the addition of calcium chloride to MP rich plasma, following incubation with Russell viper venom and phospholipid free plasma. The CT obtained by the in-house assay significantly correlated with the results obtained by flow cytometry (R2=0.87, p<0.01). Though preliminary, the in-house assay seems to be efficient, inexpensive and promising. It could definitely be utilized routinely for procoagulant MP assessment in various clinical settings.

  15. Apoptotic-cell-derived membrane microparticles and IFN-α induce an inflammatory immune response.

    Science.gov (United States)

    Niessen, Anna; Heyder, Petra; Krienke, Stefan; Blank, Norbert; Tykocinski, Lars-Oliver; Lorenz, Hanns-Martin; Schiller, Martin

    2015-07-15

    A dysregulation in the clearance of apoptotic material is considered a major pathogenetic factor for the emergence of autoimmune diseases. Apoptotic-cell-derived membrane microparticles (AdMPs), which are released from the cell surface during apoptosis, have been implicated in the pathogenesis of autoimmunity. Also of importance are cytokines, such as interferon-α (IFN-α), which is known to be a major player in patients with systemic lupus erythematosus (SLE). This study investigates the combined effect of AdMPs and IFN-α on professional phagocytes. In the presence of IFN-α, phagocytosis of AdMPs by human monocytes was significantly increased in a dose-dependent manner. The combination of AdMPs and raised IFN-α concentrations resulted in an increase in the secretion of pro-inflammatory cytokines and an upregulation of surface molecule expression involved in antigen uptake. In addition, macrophage polarisation was shifted towards a more inflammatory type of cell. The synergism between IFN-α and AdMPs seemed to be mediated by an upregulation of phosphorylated STAT1. Our results indicate that IFN-α, together with AdMPs, amplify the initiation and maintenance of inflammation. This mechanism might especially play a crucial role in disorders with a defective clearance of apoptotic material. © 2015. Published by The Company of Biologists Ltd.

  16. Early incorporation of cell-derived cholesterol into pre-beta-migrating high-density lipoprotein

    International Nuclear Information System (INIS)

    Castro, G.R.; Fielding, C.J.

    1988-01-01

    Cultures of human skin fibroblasts were labeled to high cholesterol specific activity with [ 3 H]cholesterol and incubated briefly (1-3 min) with normal human plasma. The plasma was fractionated by two-dimensional agarose-polyacrylamide gel electrophoresis and the early appearance of cholesterol label among plasma lipoproteins determined. A major part of the label at 1-min incubation was in a pre-beta-migrating apo A-I lipoprotein fraction with a molecular weight of ca. 70,000. Label was enriched about 30-fold in this fraction relative to its content of apo A-I (1-2% of total apo A-I). The proportion of label in this lipoprotein was strongly correlated with its concentration in plasma. Further incubation (2 min) in the presence of unlabeled cells demonstrated transfer of label from this fraction to a higher molecular weight pre-beta apo A-I species, to low-density lipoprotein, and to the alpha-migrating apo A-I that made up the bulk (96%) of total apo A-I in plasma. The data suggest that a significant part of cell-derived cholesterol is transferred specifically to a pre-beta-migrating lipoprotein A-I species as part of a cholesterol transport transfer sequence in plasma

  17. Potential of laryngeal muscle regeneration using induced pluripotent stem cell-derived skeletal muscle cells.

    Science.gov (United States)

    Dirja, Bayu Tirta; Yoshie, Susumu; Ikeda, Masakazu; Imaizumi, Mitsuyoshi; Nakamura, Ryosuke; Otsuki, Koshi; Nomoto, Yukio; Wada, Ikuo; Hazama, Akihiro; Omori, Koichi

    2016-01-01

    Conclusion Induced pluripotent stem (iPS) cells may be a new potential cell source for laryngeal muscle regeneration in the treatment of vocal fold atrophy after recurrent laryngeal nerve paralysis. Objectives Unilateral vocal fold paralysis can lead to degeneration, atrophy, and loss of force of the thyroarytenoid muscle. At present, there are some treatments such as thyroplasty, arytenoid adduction, and vocal fold injection. However, such treatments cannot restore reduced mass of the thyroarytenoid muscle. iPS cells have been recognized as supplying a potential resource for cell transplantation. The aim of this study was to assess the effectiveness of the use of iPS cells for the regeneration of laryngeal muscle through the evaluation of both in vitro and in vivo experiments. Methods Skeletal muscle cells were generated from tdTomato-labeled iPS cells using embryoid body formation. Differentiation into skeletal muscle cells was analyzed by gene expression and immunocytochemistry. The tdTomato-labeled iPS cell-derived skeletal muscle cells were transplanted into the left atrophied thyroarytenoid muscle. To evaluate the engraftment of these cells after transplantation, immunohistochemistry was performed. Results The tdTomato-labeled iPS cells were successfully differentiated into skeletal muscle cells through an in vitro experiment. These cells survived in the atrophied thyroarytenoid muscle after transplantation.

  18. Novel Application of Stem Cell-Derived Neurons to Evaluate the Time-and Dose-Dependent Progression of Excitotoxic Injury

    Science.gov (United States)

    2013-05-14

    3. DATES COVERED (From - To) 4. TITLE AND SUBTITLE Novel Application of Stem Cell -Derived Neurons to Evaluate the Time- and Dose-Dependent 5a...01.10.RC.021). 14. ABSTRACT See reprint. 15. SUBJECT TERMS Mouse stem cell -derived neurons, NMDA receptor, AMPA receptor, Neurotoxicity, Apoptosis...area code) 410-436-8044 Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std. Z39.18 Novel Application of Stem Cell -Derived Neurons to Evaluate the

  19. Novel paths towards neural cellular products for neurological disorders.

    Science.gov (United States)

    Daadi, Marcel M

    2011-11-01

    The prospect of using neural cells derived from stem cells or from reprogrammed adult somatic cells provides a unique opportunity in cell therapy and drug discovery for developing novel strategies for brain repair. Cell-based therapeutic approaches for treating CNS afflictions caused by disease or injury aim to promote structural repair of the injured or diseased neural tissue, an outcome currently not achieved by drug therapy. Preclinical research in animal models of various diseases or injuries report that grafts of neural cells enhance endogenous repair, provide neurotrophic support to neurons undergoing degeneration and replace lost neural cells. In recent years, the sources of neural cells for treating neurological disorders have been rapidly expanding and in addition to offering therapeutic potential, neural cell products hold promise for disease modeling and drug discovery use. Specific neural cell types have been derived from adult or fetal brain, from human embryonic stem cells, from induced pluripotent stem cells and directly transdifferentiated from adult somatic cells, such as skin cells. It is yet to be determined if the latter approach will evolve into a paradigm shift in the fields of stem cell research and regenerative medicine. These multiple sources of neural cells cover a wide spectrum of safety that needs to be balanced with efficacy to determine the viability of the cellular product. In this article, we will review novel sources of neural cells and discuss current obstacles to developing them into viable cellular products for treating neurological disorders.

  20. Poly(trimethylene carbonate) as an elastic biodegradable film for human embryonic stem cell-derived retinal pigment epithelial cells

    NARCIS (Netherlands)

    Sorkio, Anni; Haimi, Suvi; Verdoold, Vincent; Juuti-Uusitalo, Kati; Grijpma, Dirk; Skottman, Heli

    2017-01-01

    Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cell therapies show tremendous potential for the treatment of retinal degenerative diseases. A tissue engineering approach, where cells are delivered to the subretinal space on a biodegradable carrier as a sheet, shows great

  1. Poly(trimethylene carbonate) as an elastic biodegradable film for human embryonic stem cell-derived retinal pigment epithelial cells

    NARCIS (Netherlands)

    Sorkio, Anni; Haimi, Suvi; Verdoold, Vincent; Juuti-Uusitalo, Kati; Grijpma, Dirk; Skottman, Heli

    Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cell therapies show tremendous potential for the treatment of retinal degenerative diseases. A tissue engineering approach, where cells are delivered to the subretinal space on a biodegradable carrier as a sheet, shows great

  2. Feeder-cell-independent culture of the pig-embryonic-stem-cell-derived exocrine pancreatic cell line, PICM-31

    Science.gov (United States)

    The adaptation to feeder-independent growth of a pig embryonic stem cell-derived pancreatic cell line is described. The parental PICM-31 cell line, previously characterized as an exocrine pancreas cell line, was colony-cloned two times in succession resulting in the subclonal cell line, PICM-31A1. P...

  3. Induced pluripotent stem cell-derived neuron as a human model for testing environmentally induced developmental neurotoxicity

    Science.gov (United States)

    Induced pluripotent stem cell-derived neurons as a human model for testing environmentally induced developmental neurotoxicity Ingrid L. Druwe1, Timothy J. Shafer2, Kathleen Wallace2, Pablo Valdivia3 ,and William R. Mundy2. 1University of North Carolina, Curriculum in Toxicology...

  4. Endothelial cell-derived matrix promotes the metabolic functional maturation of hepatocyte via integrin-Src signalling.

    Science.gov (United States)

    Guo, Xinyue; Li, Weihong; Ma, Minghui; Lu, Xin; Zhang, Haiyan

    2017-11-01

    The extracellular matrix (ECM) microenvironment is involved in the regulation of hepatocyte phenotype and function. Recently, the cell-derived extracellular matrix has been proposed to represent the bioactive and biocompatible materials of the native ECM. Here, we show that the endothelial cell-derived matrix (EC matrix) promotes the metabolic maturation of human adipose stem cell-derived hepatocyte-like cells (hASC-HLCs) through the activation of the transcription factor forkhead box protein A2 (FOXA2) and the nuclear receptors hepatocyte nuclear factor 4 alpha (HNF4α) and pregnane X receptor (PXR). Reducing the fibronectin content in the EC matrix or silencing the expression of α5 integrin in the hASC-HLCs inhibited the effect of the EC matrix on Src phosphorylation and hepatocyte maturation. The inhibition of Src phosphorylation using the inhibitor PP2 or silencing the expression of Src in hASC-HLCs also attenuated the up-regulation of the metabolic function of hASC-HLCs in a nuclear receptor-dependent manner. These data elucidate integrin-Src signalling linking the extrinsic EC matrix signals and metabolic functional maturation of hepatocyte. This study provides a model for studying the interaction between hepatocytes and non-parenchymal cell-derived matrix. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  5. Characterization of axon formation in the embryonic stem cell-derived motoneuron.

    Science.gov (United States)

    Pan, Hung-Chuan; Wu, Ya-Ting; Shen, Shih-Cheng; Wang, Chi-Chung; Tsai, Ming-Shiun; Cheng, Fu-Chou; Lin, Shinn-Zong; Chen, Ching-Wen; Liu, Ching-San; Su, Hong-Lin

    2011-01-01

    The developing neural cell must form a highly organized architecture to properly receive and transmit nerve signals. Neural formation from embryonic stem (ES) cells provides a novel system for studying axonogenesis, which are orchestrated by polarity-regulating molecules. Here the ES-derived motoneurons, identified by HB9 promoter-driven green fluorescent protein (GFP) expression, showed characteristics of motoneuron-specific gene expression. In the majority of motoneurons, one of the bilateral neurites developed into an axon that featured with axonal markers, including Tau1, vesicle acetylcholine transporter, and synaptophysin. Interestingly, one third of the motoneurons developed bi-axonal processes but no multiple axonal GFP cell was found. The neuronal polarity-regulating proteins, including the phosphorylated AKT and ERK, were compartmentalized into both of the bilateral axonal tips. Importantly, this aberrant axon morphology was still present after the engraftment of GFP(+) neurons into the spinal cord, suggesting that even a mature neural environment fails to provide a proper niche to guide normal axon formation. These findings underscore the necessity for evaluating the morphogenesis and functionality of neurons before the clinical trials using ES or somatic stem cells.

  6. [Analysis of factors related to the number of mesenchymal stem cells derived from synovial fluid of the temporomandibular joint].

    Science.gov (United States)

    Sun, Y P; Zheng, Y H; Zhang, Z G

    2017-06-09

    Objective: To analyze related factors on the number of mesenchymal stem cells in the synovial fluid of the temporomandibular joint (TMJ) and provide an research basis for understanding of the source and biological role of mesenchymal stem cells derived from synovial fluid in TMJ. Methods: One hundred and twenty-two synovial fluid samples from 91 temporomandibular disorders (TMD) patients who visited in Department of TMJ Center, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University from March 2013 to December 2013 were collected in this study, and 6 TMJ synovial fluid samples from 6 normal volunteers who were studying in the North Campus of Sun Yat-sen University were also collected, so did their clinical information. Then the relation between the number of mesenchymal stem cells derived from synovial fluid and the health status of the joints, age of donor, disc perforation, condylar bony destruction, blood containing and visual analogue scale score of pain were investigated using Mann-Whitney U test and Spearman rank correlation test. Results: The number of mesenchymal stem cells derived from synovial fluid had no significant relation with visual analogue scale score of pain ( r= 0.041, P= 0.672), blood containing ( P= 0.063), condylar bony destruction ( P= 0.371). Linear correlation between the number of mesenchymal stem cells derived from synovial fluid and age of donor was very week ( r= 0.186, P= 0.043). The number of mesenchymal stem cells up-regulated when the joint was in a disease state ( P= 0.001). The disc perforation group had more mesenchymal stem cells in synovial fluid than without disc perforation group ( P= 0.042). Conclusions: The number of mesenchymal stem cells derived from synovial fluid in TMJ has no correlation with peripheral blood circulation and condylar bony destruction, while has close relation with soft tissue structure damage of the joint.

  7. Neural Networks

    International Nuclear Information System (INIS)

    Smith, Patrick I.

    2003-01-01

    Physicists use large detectors to measure particles created in high-energy collisions at particle accelerators. These detectors typically produce signals indicating either where ionization occurs along the path of the particle, or where energy is deposited by the particle. The data produced by these signals is fed into pattern recognition programs to try to identify what particles were produced, and to measure the energy and direction of these particles. Ideally, there are many techniques used in this pattern recognition software. One technique, neural networks, is particularly suitable for identifying what type of particle caused by a set of energy deposits. Neural networks can derive meaning from complicated or imprecise data, extract patterns, and detect trends that are too complex to be noticed by either humans or other computer related processes. To assist in the advancement of this technology, Physicists use a tool kit to experiment with several neural network techniques. The goal of this research is interface a neural network tool kit into Java Analysis Studio (JAS3), an application that allows data to be analyzed from any experiment. As the final result, a physicist will have the ability to train, test, and implement a neural network with the desired output while using JAS3 to analyze the results or output. Before an implementation of a neural network can take place, a firm understanding of what a neural network is and how it works is beneficial. A neural network is an artificial representation of the human brain that tries to simulate the learning process [5]. It is also important to think of the word artificial in that definition as computer programs that use calculations during the learning process. In short, a neural network learns by representative examples. Perhaps the easiest way to describe the way neural networks learn is to explain how the human brain functions. The human brain contains billions of neural cells that are responsible for processing

  8. Evolvable synthetic neural system

    Science.gov (United States)

    Curtis, Steven A. (Inventor)

    2009-01-01

    An evolvable synthetic neural system includes an evolvable neural interface operably coupled to at least one neural basis function. Each neural basis function includes an evolvable neural interface operably coupled to a heuristic neural system to perform high-level functions and an autonomic neural system to perform low-level functions. In some embodiments, the evolvable synthetic neural system is operably coupled to one or more evolvable synthetic neural systems in a hierarchy.

  9. Comparison of immunodulatory properties of dental pulp stem cells derived from healthy and inflamed teeth.

    Science.gov (United States)

    Yazid, Farinawati Binti; Gnanasegaran, Nareshwaran; Kunasekaran, Wijenthiran; Govindasamy, Vijayendran; Musa, Sabri

    2014-12-01

    The aim of this study was to investigate the immunodulatory properties of dental pulp stem cells derived from healthy (SCD) and inflamed pulp deciduous (SCDIP) tissues. The overall hypothesis is that SCDIP possess equal immune properties with SCD and could be used as an alternative tissue source in regenerative medicine. An intra-oral examination was carried out to assess the status of the pulp tissues and group them according to healthy or inflamed. Primary cells were established from these groups, and basic mesenchymal stem cells (MSC) characterizations were conducted. The expression of human leukocyte antigen (HLA), namely HLA-G, HLA-DR, and HLA-ABC were examined in both cell lines using flow cytometry. We further compared the immunosuppressive effects of SCD and SCDIP on phytohemagglutinin-induced T cell proliferation. Supernatants were tested for cytokine profiling using multiplex array. While SCD exhibited typical MSC characteristics, SCDIP on the other hand, did not. Compared with SCDIP, SCD effectively suppresses mitogen-induced T cells proliferation in a dose-dependent manner, as well as express a higher percentage of HLA-ABC and HLA-G. In addition, levels of several cytokines, such as TNF-α, TNF-β, and IL-2, were drastically suppressed in SCD than SCDIP. Furthermore, a high level of IL-10, an important anti-inflammatory cytokine, was present in SCD compared with SCDIP. These findings suggest that SCDIP is highly dysfunctional in terms of their stemness and immunomodulatory properties. SCDIP is not a viable therapeutic cell source especially when used in graft versus host disease (GvHD) and organ rejection.

  10. Generation of induced pluripotent stem cell-derived mice by reprogramming of a mature NKT cell.

    Science.gov (United States)

    Ren, Yue; Dashtsoodol, Nyambayar; Watarai, Hiroshi; Koseki, Haruhiko; Quan, Chengshi; Taniguchi, Masaru

    2014-10-01

    NKT cells are characterized by their expression of an NKT-cell-specific invariant antigen-receptor α chain encoded by Vα14Jα18 gene segments. These NKT cells bridge the innate and acquired immune systems to mediate effective and augmented responses; however, the limited number of NKT cells in vivo hampers their analysis. Here, two lines of induced pluripotent stem cell-derived mice (NKT-iPSC-derived mice) were generated by reprogramming of mature NKT cells, where one harbors both rearranged Vα14Jα18 and Vβ7 genes and the other carries rearranged Vα14Jα18 on both alleles but germline Vβ loci. The analysis of NKT-iPSC-derived mice showed a significant increase in NKT cell numbers with relatively normal frequencies of functional subsets, but significantly enhanced in some cases, and acquired functional NKT cell maturation in peripheral lymphoid organs. NKT-iPSC-derived mice also showed normal development of other immune cells except for the absence of γδT cells and disturbed development of conventional CD4 αβT cells. These results suggest that the NKT-iPSC-derived mice are a better model for NKT cell development and function study rather than transgenic mouse models reported previously and also that the presence of a pre-rearranged Vα14Jα18 in the natural chromosomal context favors the developmental fate of NKT cells. © The Author 2014. Published by Oxford University Press on behalf of The Japanese Society for Immunology.

  11. Slow conduction in mixed cultured strands of primary ventricular cells and stem cell-derived cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Jan Pavel Kucera

    2015-09-01

    Full Text Available Modern concepts for the treatment of myocardial diseases focus on novel cell therapeutic strategies involving stem cell-derived cardiomyocytes (SCMs. However, functional integration of SCMs requires similar electrophysiological properties as primary cardiomyocytes (PCMs and the ability to establish intercellular connections with host myocytes in order to contribute to the electrical and mechanical activity of the heart. The aim of this project was to investigate the properties of cardiac conduction in a co-culture approach using SCMs and PCMs in cultured cell strands. Murine embryonic SCMs were pooled with fetal ventricular cells and seeded in predefined proportions on microelectrode arrays to form patterned strands of mixed cells. Conduction velocity (CV was measured during steady state pacing. SCM excitability was estimated from action potentials measured in single cells using the patch clamp technique. Experiments were complemented with computer simulations of conduction using a detailed model of cellular architecture in mixed cell strands.CV was significantly lower in strands composed purely of SCMs (5.5±1.5 cm/s, n=11 as compared to PCMs (34.9±2.9 cm/s, n=21 at similar refractoriness (100% SCMs: 122±25 ms, n=9; 100% PCMs: 139±67 ms, n=14. In mixed strands combining both cell types, CV was higher than in pure SCMs strands, but always lower than in 100% PCM strands. Computer simulations demonstrated that both intercellular coupling and electrical excitability limit CV.These data provide evidence that in cultures of murine ventricular cardiomyocytes, SCMs cannot restore CV to control levels resulting in slow conduction, which may lead to reentry circuits and arrhythmias.

  12. Stromal cell-derived factor 1 regulates the actin organization of chondrocytes and chondrocyte hypertrophy.

    Directory of Open Access Journals (Sweden)

    Koichi Murata

    Full Text Available Stromal cell-derived factor 1 (SDF-1/CXCL12/PBSF plays important roles in the biological and physiological functions of haematopoietic and mesenchymal stem cells. This chemokine regulates the formation of multiple organ systems during embryogenesis. However, its roles in skeletal development remain unclear. Here we investigated the roles of SDF-1 in chondrocyte differentiation. We demonstrated that SDF-1 protein was expressed at pre-hypertrophic and hypertrophic chondrocytes in the newly formed endochondral callus of rib fracture as well as in the growth plate of normal mouse tibia by immunohistochemical analysis. Using SDF-1(-/- mouse embryo, we histologically showed that the total length of the whole humeri of SDF-1(-/- mice was significantly shorter than that of wild-type mice, which was contributed mainly by shorter hypertrophic and calcified zones in SDF-1(-/- mice. Actin cytoskeleton of hypertrophic chondrocytes in SDF-1(-/- mouse humeri showed less F-actin and rounder shape than that of wild-type mice. Primary chondrocytes from SDF-1(-/- mice showed the enhanced formation of philopodia and loss of F-actin. The administration of SDF-1 to primary chondrocytes of wild-type mice and SDF-1(-/- mice promoted the formation of actin stress fibers. Organ culture of embryonic metatarsals from SDF-1(-/- mice showed the growth delay, which was recovered by an exogenous administration of SDF-1. mRNA expression of type X collagen in metatarsals and in primary chondrocytes of SDF-1(-/- mouse embryo was down-regulated while the administration of SDF-1 to metatarsals recovered. These data suggests that SDF-1 regulates the actin organization and stimulates bone growth by mediating chondrocyte hypertrophy.

  13. Stromal cell-derived factor 1 regulates the actin organization of chondrocytes and chondrocyte hypertrophy.

    Science.gov (United States)

    Murata, Koichi; Kitaori, Toshiyuki; Oishi, Shinya; Watanabe, Naoki; Yoshitomi, Hiroyuki; Tanida, Shimei; Ishikawa, Masahiro; Kasahara, Takashi; Shibuya, Hideyuki; Fujii, Nobutaka; Nagasawa, Takashi; Nakamura, Takashi; Ito, Hiromu

    2012-01-01

    Stromal cell-derived factor 1 (SDF-1/CXCL12/PBSF) plays important roles in the biological and physiological functions of haematopoietic and mesenchymal stem cells. This chemokine regulates the formation of multiple organ systems during embryogenesis. However, its roles in skeletal development remain unclear. Here we investigated the roles of SDF-1 in chondrocyte differentiation. We demonstrated that SDF-1 protein was expressed at pre-hypertrophic and hypertrophic chondrocytes in the newly formed endochondral callus of rib fracture as well as in the growth plate of normal mouse tibia by immunohistochemical analysis. Using SDF-1(-/-) mouse embryo, we histologically showed that the total length of the whole humeri of SDF-1(-/-) mice was significantly shorter than that of wild-type mice, which was contributed mainly by shorter hypertrophic and calcified zones in SDF-1(-/-) mice. Actin cytoskeleton of hypertrophic chondrocytes in SDF-1(-/-) mouse humeri showed less F-actin and rounder shape than that of wild-type mice. Primary chondrocytes from SDF-1(-/-) mice showed the enhanced formation of philopodia and loss of F-actin. The administration of SDF-1 to primary chondrocytes of wild-type mice and SDF-1(-/-) mice promoted the formation of actin stress fibers. Organ culture of embryonic metatarsals from SDF-1(-/-) mice showed the growth delay, which was recovered by an exogenous administration of SDF-1. mRNA expression of type X collagen in metatarsals and in primary chondrocytes of SDF-1(-/-) mouse embryo was down-regulated while the administration of SDF-1 to metatarsals recovered. These data suggests that SDF-1 regulates the actin organization and stimulates bone growth by mediating chondrocyte hypertrophy.

  14. Modeling chemotherapeutic neurotoxicity with human induced pluripotent stem cell-derived neuronal cells.

    Directory of Open Access Journals (Sweden)

    Heather E Wheeler

    Full Text Available There are no effective agents to prevent or treat chemotherapy-induced peripheral neuropathy (CIPN, the most common non-hematologic toxicity of chemotherapy. Therefore, we sought to evaluate the utility of human neuron-like cells derived from induced pluripotent stem cells (iPSCs as a means to study CIPN. We used high content imaging measurements of neurite outgrowth phenotypes to compare the changes that occur to iPSC-derived neuronal cells among drugs and among individuals in response to several classes of chemotherapeutics. Upon treatment of these neuronal cells with the neurotoxic drug paclitaxel, vincristine or cisplatin, we identified significant differences in five morphological phenotypes among drugs, including total outgrowth, mean/median/maximum process length, and mean outgrowth intensity (P < 0.05. The differences in damage among drugs reflect differences in their mechanisms of action and clinical CIPN manifestations. We show the potential of the model for gene perturbation studies by demonstrating decreased expression of TUBB2A results in significantly increased sensitivity of neurons to paclitaxel (0.23 ± 0.06 decrease in total neurite outgrowth, P = 0.011. The variance in several neurite outgrowth and apoptotic phenotypes upon treatment with one of the neurotoxic drugs is significantly greater between than within neurons derived from four different individuals (P < 0.05, demonstrating the potential of iPSC-derived neurons as a genetically diverse model for CIPN. The human neuron model will allow both for mechanistic studies of specific genes and genetic variants discovered in clinical studies and for screening of new drugs to prevent or treat CIPN.

  15. Functional cardiotoxicity assessment of cosmetic compounds using human-induced pluripotent stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Chaudhari, Umesh; Nemade, Harshal; Sureshkumar, Poornima; Vinken, Mathieu; Ates, Gamze; Rogiers, Vera; Hescheler, Jürgen; Hengstler, Jan Georg; Sachinidis, Agapios

    2018-01-01

    There is a large demand of a human relevant in vitro test system suitable for assessing the cardiotoxic potential of cosmetic ingredients and other chemicals. Using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we have already established an in vitro cardiotoxicity assay and identified genomic biomarkers of anthracycline-induced cardiotoxicity in our previous work. Here, five cosmetic ingredients were studied by the new hiPSC-CMs test; kojic acid (KJA), triclosan (TS), triclocarban (TCC), 2,7-naphthalenediol (NPT), and basic red 51 (BR51) based on cytotoxicity as well as ATP assays, beating rate, and genomic biomarkers to determine the lowest observed effect concentration (LOEC) and no observed effect concentration (NOEC). The LOEC for beating rate were 400, 10, 3, >400, and 3 µM for KJA, TS, TCC, NPT, and BR51, respectively. The corresponding concentrations for cytotoxicity or ATP depletion were similar, with the exception of TS and TCC, where the cardiomyocyte-beating assay showed positive results at non-cytotoxic concentrations. Functional analysis also showed that the individual compounds caused different effects on hiPSC-CMs. While exposure to KJA, TS, TCC, and BR51 induced significant arrhythmic beating, NPT slightly decreased cell viability, but did not influence beating. Gene expression studies showed that TS and NPT caused down-regulation of cytoskeletal and cardiac ion homeostasis genes. Moreover, TS and NPT deregulated genomic biomarkers known to be affected also by anthracyclines. The present study demonstrates that hiPSC-CMs can be used to determine LOECs and NOECs in vitro, which can be compared to human blood concentrations to determine margins of exposure. Our in vitro assay, which so far has been tested with several anthracyclines and cosmetics, still requires validation by larger numbers of positive and negative controls, before it can be recommended for routine analysis.

  16. Targeting eradication of malignant cells derived from human bone marrow mesenchymal stromal cells

    International Nuclear Information System (INIS)

    Yang, Yingbin; Cai, Shaoxi; Yang, Li; Yu, Shuhui; Jiang, Jiahuan; Yan, Xiaoqing; Zhang, Haoxing; Liu, Lan; Liu, Qun; Du, Jun; Cai, Shaohui; Sung, K.L. Paul

    2010-01-01

    Human bone marrow mesenchymal stromal cells (hBMSC) have been shown to participate in malignant transformation. However, hampered by the low frequency of malignant transformation of hBMSC, we do not yet know how to prevent malignant transformation of implanted hBMSC. In this study, in order to establish a model for the eradication of hBMSC-derived malignant cells, a gene fusion consisting of a human telomerase (hTERT) promoter modified with both c-Myc and myeloid zinc finger protein2 (MZF-2) binding elements and followed by the E. coli cytosine deaminase (CD) and luciferase genes was stably transferred into hBMSC via lentiviral transduction; n-phosphonacelyl-L-aspartic acid (PALA) selection was used to generate malignant cell colonies derived from transduced hBMSC after treatment with the carcinogenic reagent BPDE. Cells that were amplified after PALA selection were used for transplantation and 5-FC pro-drug cytotoxicity tests. The results showed that PALA-resistant malignant cells could be generated from hBMSC co-induced with lentiviral transduction and treatment with Benzo(a)pyrene Diol Epoxide (BPDE); the modification of c-Myc and MZF-2 binding elements could remarkably enhance the transcriptional activities of the hTERT promoter in malignant cells, whereas transcriptional activity was depressed in normal hBMSC; malignant cells stably expressing CD under the control of the modified hTERT promoter could be eliminated by 5-FC administration. This study has provided a method for targeted eradication of malignant cells derived from hBMSC.

  17. Proarrhythmia risk prediction using human induced pluripotent stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Yamazaki, Daiju; Kitaguchi, Takashi; Ishimura, Masakazu; Taniguchi, Tomohiko; Yamanishi, Atsuhiro; Saji, Daisuke; Takahashi, Etsushi; Oguchi, Masao; Moriyama, Yuta; Maeda, Sanae; Miyamoto, Kaori; Morimura, Kaoru; Ohnaka, Hiroki; Tashibu, Hiroyuki; Sekino, Yuko; Miyamoto, Norimasa; Kanda, Yasunari

    2018-04-01

    Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are expected to become a useful tool for proarrhythmia risk prediction in the non-clinical drug development phase. Several features including electrophysiological properties, ion channel expression profile and drug responses were investigated using commercially available hiPSC-CMs, such as iCell-CMs and Cor.4U-CMs. Although drug-induced arrhythmia has been extensively examined by microelectrode array (MEA) assays in iCell-CMs, it has not been fully understood an availability of Cor.4U-CMs for proarrhythmia risk. Here, we evaluated the predictivity of proarrhythmia risk using Cor.4U-CMs. MEA assay revealed linear regression between inter-spike interval and field potential duration (FPD). The hERG inhibitor E-4031 induced reverse-use dependent FPD prolongation. We next evaluated the proarrhythmia risk prediction by a two-dimensional map, which we have previously proposed. We determined the relative torsade de pointes risk score, based on the extent of FPD with Fridericia's correction (FPDcF) change and early afterdepolarization occurrence, and calculated the margins normalized to free effective therapeutic plasma concentrations. The drugs were classified into three risk groups using the two-dimensional map. This risk-categorization system showed high concordance with the torsadogenic information obtained by a public database CredibleMeds. Taken together, these results indicate that Cor.4U-CMs can be used for drug-induced proarrhythmia risk prediction. Copyright © 2018 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  18. Knockout of endothelial cell-derived endothelin-1 attenuates skin fibrosis but accelerates cutaneous wound healing.

    Directory of Open Access Journals (Sweden)

    Katsunari Makino

    Full Text Available Endothelin (ET-1 is known for the most potent vasoconstrictive peptide that is released mainly from endothelial cells. Several studies have reported ET-1 signaling is involved in the process of wound healing or fibrosis as well as vasodilation. However, little is known about the role of ET-1 in these processes. To clarify its mechanism, we compared skin fibrogenesis and wound repair between vascular endothelial cell-specific ET-1 knockout mice and their wild-type littermates. Bleomycin-injected fibrotic skin of the knockout mice showed significantly decreased skin thickness and collagen content compared to that of wild-type mice, indicating that bleomycin-induced skin fibrosis is attenuated in the knockout mice. The mRNA levels of transforming growth factor (TGF-β were decreased in the bleomycin-treated skin of ET-1 knockout mice. On the other hand, skin wound healing was accelerated in ET-1 knockout mice, which was indicated by earlier granulation tissue reduction and re-epithelialization in these mice. The mRNA levels of TGF-β, tumor necrosis factor (TNF-α and connective tissue growth factor (CTGF were reduced in the wound of ET-1 knockout mice. In endothelial ET-1 knockout mouse, the expression of TNF-α, CTGF and TGF-β was down-regulated. Bosentan, an antagonist of dual ET receptors, is known to attenuate skin fibrosis and accelerate wound healing in systemic sclerosis, and such contradictory effect may be mediated by above molecules. The endothelial cell-derived ET-1 is the potent therapeutic target in fibrosis or wound healing, and investigations of the overall regulatory mechanisms of these pathological conditions by ET-1 may lead to a new therapeutic approach.

  19. Usefulness of cardiotoxicity assessment using calcium transient in human induced pluripotent stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Watanabe, Hitoshi; Honda, Yayoi; Deguchi, Jiro; Yamada, Toru; Bando, Kiyoko

    2017-01-01

    Monitoring dramatic changes in intracellular calcium ion levels during cardiac contraction and relaxation, known as calcium transient, in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) would be an attractive strategy for assessing compounds on cardiac contractility. In addition, as arrhythmogenic compounds are known to induce characteristic waveform changes in hiPSC-CMs, it is expected that calcium transient would allow evaluation of not only compound-induced effects on cardiac contractility, but also compound arrhythmogenic potential. Using a combination of calcium transient in hiPSC-CMs and a fast kinetic fluorescence imaging detection system, we examined in this study changes in calcium transient waveforms induced by a series of 17 compounds that include positive/negative inotropic agents as well as cardiac ion channel activators/inhibitors. We found that all positive inotropic compounds induced an increase in peak frequency and/or peak amplitude. The effects of a negative inotropic compound could clearly be detected in the presence of a β-adrenergic receptor agonist. Furthermore, most arrhythmogenic compounds raised the ratio of peak decay time to peak rise time (D/R ratio) in calcium transient waveforms. Compound concentrations at which these parameters exceeded cutoff values correlated well with systemic exposure levels at which arrhythmias were reported to be evoked. In conclusion, we believe that peak analysis of calcium transient and determination of D/R ratio are reliable methods for assessing compounds' cardiac contractility and arrhythmogenic potential, respectively. Using these approaches would allow selection of compounds with low cardiotoxic potential at the early stage of drug discovery.

  20. Targeting eradication of malignant cells derived from human bone marrow mesenchymal stromal cells

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Yingbin [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); School of Life Science, Southwest University, Chongqing 400715 (China); Cai, Shaoxi, E-mail: sxcai@cqu.edu.cn [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); Yang, Li [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); College of Pharmacy, Jinan University, Guangzhou 510632 (China); Yu, Shuhui [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); Library of Southwest University, Chongqing 400715 (China); Jiang, Jiahuan; Yan, Xiaoqing [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); Zhang, Haoxing [School of Life Science, Southwest University, Chongqing 400715 (China); Liu, Lan [Department of Laboratory of Medicine, Children' s Hospital of Chongqin Medical University, Chongqing 400014 (China); Liu, Qun [College of Life Science and Technology, Southwest University for Nationalities, Chengdu 610041 (China); Du, Jun [Center of Microbiology, Biochemistry, and Pharmacology, School of Pharmaceutical Science, Sun Yat-Sen University, Guangzhou 510080 (China); Cai, Shaohui [College of Pharmacy, Jinan University, Guangzhou 510632 (China); Sung, K.L. Paul [Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College, Chongqing University, Chongqing 400044 (China); Departments of Orthopaedic Surgery and Bioengineering, University of California, SD 0412 (United States)

    2010-12-10

    Human bone marrow mesenchymal stromal cells (hBMSC) have been shown to participate in malignant transformation. However, hampered by the low frequency of malignant transformation of hBMSC, we do not yet know how to prevent malignant transformation of implanted hBMSC. In this study, in order to establish a model for the eradication of hBMSC-derived malignant cells, a gene fusion consisting of a human telomerase (hTERT) promoter modified with both c-Myc and myeloid zinc finger protein2 (MZF-2) binding elements and followed by the E. coli cytosine deaminase (CD) and luciferase genes was stably transferred into hBMSC via lentiviral transduction; n-phosphonacelyl-L-aspartic acid (PALA) selection was used to generate malignant cell colonies derived from transduced hBMSC after treatment with the carcinogenic reagent BPDE. Cells that were amplified after PALA selection were used for transplantation and 5-FC pro-drug cytotoxicity tests. The results showed that PALA-resistant malignant cells could be generated from hBMSC co-induced with lentiviral transduction and treatment with Benzo(a)pyrene Diol Epoxide (BPDE); the modification of c-Myc and MZF-2 binding elements could remarkably enhance the transcriptional activities of the hTERT promoter in malignant cells, whereas transcriptional activity was depressed in normal hBMSC; malignant cells stably expressing CD under the control of the modified hTERT promoter could be eliminated by 5-FC administration. This study has provided a method for targeted eradication of malignant cells derived from hBMSC.

  1. Profiling ethanol-targeted transcription factors in human carcinoma cell-derived embryoid bodies.

    Science.gov (United States)

    Mandal, Chanchal; Halder, Debasish; Chai, Jin Choul; Lee, Young Seek; Jung, Kyoung Hwa; Chai, Young Gyu

    2016-01-15

    Fetal alcohol spectrum disorder is a collective term that represents fetal abnormalities associated with maternal alcohol consumption. Prenatal alcohol exposure and related anomalies are well characterized, but the molecular mechanism behind this phenomenon is not yet understood. Few insights have been gained from genetic and epigenetic studies of fetal alcohol spectrum disorder. Our aim was to profile the important molecular regulators of ethanol-related alterations of the genome. For this purpose, we have analyzed the gene expression pattern of human carcinoma cell-derived embryoid bodies in the absence or presence of ethanol. A cDNA microarray analysis was used to profile mRNA expression in embryoid bodies at day 7 with or without ethanol treatment. A total of 493 differentially expressed genes were identified in response to 50 mM ethanol exposure. Of these, 111 genes were up-regulated, and 382 were down-regulated. Gene ontology term enrichment analysis revealed that these genes are involved in important biological processes: neurological system processes, cognition, behavior, sensory perception of smell, taste and chemical stimuli and synaptic transmission. Similarly, the enrichment of disease-related genes included relevant categories such as neurological diseases, developmental disorders, skeletal and muscular disorders, and connective tissue disorders. Furthermore, we have identified a group of 26 genes that encode transcription factors. We validated the relative gene expression of several transcription factors using quantitative real time PCR. We hope that our study substantially contributes to the understanding of the molecular mechanisms underlying the pathology of alcohol-mediated anomalies and facilitates further research. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Liver cell-derived microparticles activate hedgehog signaling and alter gene expression in hepatic endothelial cells.

    Science.gov (United States)

    Witek, Rafal P; Yang, Liu; Liu, Renshui; Jung, Youngmi; Omenetti, Alessia; Syn, Wing-Kin; Choi, Steve S; Cheong, Yeiwon; Fearing, Caitlin M; Agboola, Kolade M; Chen, Wei; Diehl, Anna Mae

    2009-01-01

    Angiogenesis contributes to vascular remodeling during cirrhosis. In cirrhotic livers, cholangiocytes, and myofibroblastic hepatic stellate cells (MF-HSC) produce Hedgehog (Hh) ligands. During embryogenesis Hh ligands are released from ligand-producing cells in microparticles and activate Hh signaling in endothelial cells. We studied whether adult liver cell-derived microparticles contain Hh ligands that alter hepatic sinusoidal endothelial cells (SEC). MF-HSC and cholangiocytes were exposed to platelet-derived growth factor to induce Hh ligands; microparticles were isolated from medium, analyzed by transmission electron microscopy and immunoblots, and applied to Hh-reporter-containing cells. Microparticles were obtained from serum and bile of rats after bile duct ligation (BDL) or sham surgery and applied to normal primary liver SEC with or without cyclopamine, an Hh signaling inhibitor. Effects on SEC gene expression were evaluated by quantitative reverse-transcription polymerase chain reaction and immunoblotting. Hh target gene expression and SEC activation markers were compared in primary SEC and in liver sections from healthy and BDL rats. Platelet-derived growth factor-treated MF-HSC and cholangiocytes released exosome-enriched microparticles containing biologically-active Hh ligands. BDL increased release of Hh-containing exosome-enriched microparticles into plasma and bile. Transmission electron microscopy and immunoblots revealed similarities among microparticles from all sources; all microparticles induced similar Hh-dependent changes in SEC gene expression. SEC from healthy livers did not express Hh target genes or activation markers, but both were up-regulated in SEC after BDL. Hh-containing exosome-enriched microparticles released from liver cells alter hepatic SEC gene expression, suggesting a novel mechanism for cirrhotic vasculopathy.

  3. Apheresis platelet concentrates contain platelet-derived and endothelial cell-derived microparticles.

    Science.gov (United States)

    Rank, A; Nieuwland, R; Liebhardt, S; Iberer, M; Grützner, S; Toth, B; Pihusch, R

    2011-02-01

    Microparticles (MP) are membrane vesicles with thrombogenic and immunomodulatory properties. We determined MP subgroups from resting platelets, activated platelets and endothelial cells in donors and apheresis platelet concentrates (PC). MP were double stained with annexin V and CD61 (platelet-derived MP; PMP), P-selectin or CD63 (MP from activated platelets) and CD144 plus E-selectin (endothelial cell-derived MP; EMP) and detected by flow cytometry in platelet donors (n=36) and apheresis PC (n=11; Trima™). PC contained MP, mainly from resting platelets [93% (90-95)], and minor fractions of PMP from activated platelets [P-selectin(+) or CD63(+); 4·8% (3·2-7·7) and 2·6% (2·0-4·0)]. Compared to donors, levels of annexin V+ MP, PMP, P-selectin(+) and CD63(+) MP were 1·7-, 2·3-, 8·6- and 3·1-fold higher in PC (all P<0·05). During storage (1-5 days), levels of annexin V+ MP and PMP did not increase, although small increases in the fraction of P-selectin(+) or CD63(+) MP occurred (both P<0·05). PC also contained EMP, which were 2·6- to 3·7-fold enriched in PC compared to donors (P<0·05). Transfusion of apheresis PC also results in transfusion of HLA-carrying PMP and EMP. This might counteract the aim of reducing transfused HLA load by leucodepletion. The increases in PMP exposing P-selectin or CD63 reflect mild platelet activation during storage. We conclude that in leucodepleted platelet apheresis using fluidized particle bed technology, MP are harvested mainly from the donor by apheresis. Improvement in apheresis technology might reduce MP load. © 2010 The Author(s). Vox Sanguinis © 2010 International Society of Blood Transfusion.

  4. A novel broadband impedance method for detection of cell-derived microparticles.

    Science.gov (United States)

    Lvovich, Vadim; Srikanthan, Sowmya; Silverstein, Roy L

    2010-10-15

    A novel label-free method is presented to detect and quantify cell-derived microparticles (MPs) by the electrochemical potential-modulated electrochemical impedance spectroscopy (EIS). MPs are present in elevated concentrations during pathological conditions and play a major role in the establishment and pathogenesis of many diseases. Considering this, accurate detection and quantification of MPs is very important in clinical diagnostics and therapeutics. A combination of bulk solution electrokinetic sorting and interfacial impedance responses allows achieving detection limits as low as several MPs per μL. By fitting resulting EIS spectra with an equivalent electrical circuit, the bulk solution electrokinetic and interfacial impedance responses were characterized. In the bulk solution two major relaxations were prominent-β-relaxation in low MHz region due to the MP capacitive membrane bridging, and α-relaxation at ∼10 kHz due to counter ions diffusion. At low frequencies (10-0.1 Hz) at electrochemical potentials exceeding -100 mV, a facile interfacial Faradaic process of oxidation in MPs coupled with diffusion and non-Faradaic double layer charging dominate, probably due to oxidation of phospholipids and/or proteins on the MP surface and MP lysis. Buffer influence on the MP detection demonstrated that a relatively low conductivity Tyrode's buffer background solution is preferential for the MP electrokinetic separation and characterization. This study also demonstrated that standard laboratory methods such as flow cytometry underestimate MP concentrations, especially those with smaller average sizes, by as much as a factor of 2-40. Copyright © 2010 Elsevier B.V. All rights reserved.

  5. Measurement of circulating cell-derived microparticles by flow cytometry: sources of variability within the assay.

    Science.gov (United States)

    Ayers, Lisa; Kohler, Malcolm; Harrison, Paul; Sargent, Ian; Dragovic, Rebecca; Schaap, Marianne; Nieuwland, Rienk; Brooks, Susan A; Ferry, Berne

    2011-04-01

    Circulating cell-derived microparticles (MPs) have been implicated in several disease processes and elevated levels are found in many pathological conditions. The detection and accurate measurement of MPs, although attracting widespread interest, is hampered by a lack of standardisation. The aim of this study was to establish a reliable flow cytometric assay to measure distinct subtypes of MPs in disease and to identify any significant causes of variability in MP quantification. Circulating MPs within plasma were identified by their phenotype (platelet, endothelial, leukocyte and annexin-V positivity (AnnV+). The influence of key variables (i.e. time between venepuncture and centrifugation, washing steps, the number of centrifugation steps, freezing/long-term storage and temperature of thawing) on MP measurement were investigated. Increasing time between venepuncture and centrifugation leads to increased MP levels. Washing samples results in decreased AnnV+MPs (P=0.002) and platelet-derived MPs (PMPs) (P=0.002). Double centrifugation of MPs prior to freezing decreases numbers of AnnV+MPs (P=0.0004) and PMPs (P=0.0004). A single freeze thaw cycle of samples led to an increase in AnnV+MPs (P=0.0020) and PMPs (P=0.0039). Long-term storage of MP samples at -80° resulted in decreased MP levels. This study found that minor protocol changes significantly affected MP levels. This is one of the first studies attempting to standardise a method for obtaining and measuring circulating MPs. Standardisation will be essential for successful development of MP technologies, allowing direct comparison of results between studies and leading to a greater understanding of MPs in disease. Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.

  6. Flow rate calibration to determine cell-derived microparticles and homogeneity of blood components.

    Science.gov (United States)

    Noulsri, Egarit; Lerdwana, Surada; Kittisares, Kulvara; Palasuwan, Attakorn; Palasuwan, Duangdao

    2017-08-01

    Cell-derived microparticles (MPs) are currently of great interest to screening transfusion donors and blood components. However, the current approach to counting MPs is not affordable for routine laboratory use due to its high cost. The current study aimed to investigate the potential use of flow-rate calibration for counting MPs in whole blood, packed red blood cells (PRBCs), and platelet concentrates (PCs). The accuracy of flow-rate calibration was investigated by comparing the platelet counts of an automated counter and a flow-rate calibrator. The concentration of MPs and their origins in whole blood (n=100), PRBCs (n=100), and PCs (n=92) were determined using a FACSCalibur. The MPs' fold-changes were calculated to assess the homogeneity of the blood components. Comparing the platelet counts conducted by automated counting and flow-rate calibration showed an r 2 of 0.6 (y=0.69x+97,620). The CVs of the within-run and between-run variations of flow-rate calibration were 8.2% and 12.1%, respectively. The Bland-Altman plot showed a mean bias of -31,142platelets/μl. MP enumeration revealed both the difference in MP levels and their origins in whole blood, PRBCs, and PCs. Screening the blood components demonstrated high heterogeneity of the MP levels in PCs when compared to whole blood and PRBCs. The results of the present study suggest the accuracy and precision of flow-rate calibration for enumerating MPs. This flow-rate approach is affordable for assessing the homogeneity of MPs in blood components in routine laboratory practice. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Red blood cell-derived microparticles isolated from blood units initiate and propagate thrombin generation.

    Science.gov (United States)

    Rubin, Olivier; Delobel, Julien; Prudent, Michel; Lion, Niels; Kohl, Kid; Tucker, Erik I; Tissot, Jean-Daniel; Angelillo-Scherrer, Anne

    2013-08-01

    Red blood cell-derived microparticles (RMPs) are small phospholipid vesicles shed from RBCs in blood units, where they accumulate during storage. Because microparticles are bioactive, it could be suggested that RMPs are mediators of posttransfusion complications or, on the contrary, constitute a potential hemostatic agent. This study was performed to establish the impact on coagulation of RMPs isolated from blood units. Using calibrated automated thrombography, we investigated whether RMPs affect thrombin generation (TG) in plasma. We found that RMPs were not only able to increase TG in plasma in the presence of a low exogenous tissue factor (TF) concentration, but also to initiate TG in plasma in absence of exogenous TF. TG induced by RMPs in the absence of exogenous TF was neither affected by the presence of blocking anti-TF nor by the absence of Factor (F)VII. It was significantly reduced in plasma deficient in FVIII or F IX and abolished in FII-, FV-, FX-, or FXI-deficient plasma. TG was also totally abolished when anti-XI 01A6 was added in the sample. Finally, neither Western blotting, flow cytometry, nor immunogold labeling allowed the detection of traces of TF antigen. In addition, RMPs did not comprise polyphosphate, an important modulator of coagulation. Taken together, our data show that RMPs have FXI-dependent procoagulant properties and are able to initiate and propagate TG. The anionic surface of RMPs might be the site of FXI-mediated TG amplification and intrinsic tenase and prothrombinase complex assembly. © 2012 American Association of Blood Banks.

  8. Non-coding RNAs in Mesenchymal Stem Cell-Derived Extracellular Vesicles: Deciphering Regulatory Roles in Stem Cell Potency, Inflammatory Resolve, and Tissue Regeneration

    Directory of Open Access Journals (Sweden)

    Farah Fatima

    2017-10-01

    Full Text Available Extracellular vesicles (EVs are heterogeneous populations of nano- and micro-sized vesicles secreted by various cell types. There is mounting evidence that EVs have widespread roles in transporting proteins, lipids, and nucleic acids between cells and serve as mediators of intercellular communication. EVs secreted from stem cells could function as paracrine factors, and appear to mimic and recapitulate several features of their secreting cells. EV-mediated transport of regulatory RNAs provides a novel source of trans-regulation between cells. As such, stem cells have evolved unique forms of paracrine mechanisms for recapitulating their potencies with specialized functions by transporting non-coding RNAs (ncRNAs via EVs. This includes the dissemination of stem cell-derived EV-ncRNAs and their regulatory effects elicited in differentiation, self-renewal, pluripotency, and the induction of reparative programs. Here, we summarize and discuss the therapeutic effects of mesenchymal stem cell-derived EV-ncRNAs in the induction of intrinsic regenerative programs elicited through regulating several mechanisms. Among them, most noticeable are the EV-mediated enrichment of ncRNAs at the injury sites contributing the regulation of matrix remodeling, epithelial mesenchymal transitions, and attraction of fibroblasts. Additionally, we emphasize EV-mediated transmission of anti-inflammatory RNAs from stem cells to injury site that potentially orchestrate the resolution of the inflammatory responses and immune alleviation to better facilitate healing processes. Collectively, this knowledge indicates a high value and potential of EV-mediated RNA-based therapeutic approaches in regenerative medicine.

  9. Implantable Neural Interfaces for Sharks

    Science.gov (United States)

    2007-05-01

    technology for recording and stimulating from the auditory and olfactory sensory nervous systems of the awake, swimming nurse shark , G. cirratum (Figures...overlay of the central nervous system of the nurse shark on a horizontal MR image. Implantable Neural Interfaces for Sharks ...Neural Interfaces for Characterizing Population Responses to Odorants and Electrical Stimuli in the Nurse Shark , Ginglymostoma cirratum.” AChemS Abs

  10. Human mesenchymal stem cells derived from limb bud can differentiate into all three embryonic germ layers lineages.

    Science.gov (United States)

    Jiao, Fei; Wang, Juan; Dong, Zhao-Lun; Wu, Min-Juan; Zhao, Ting-Bao; Li, Dan-Dan; Wang, Xin

    2012-08-01

    Mesenchymal stem cells (MSCs) have been isolated from many sources, including adults and fetuses. Previous studies have demonstrated that, compared with their adult counterpart, fetal MSCs with several remarkable advantages may be a better resource for clinical applications. In this study, we successfully isolated a rapidly proliferating cell population from limb bud of aborted fetus and termed them "human limb bud-derived mesenchymal stem cells" (hLB-MSCs). Characteristics of their morphology, phenotype, cell cycle, and differentiation properties were analyzed. These adherent cell populations have a typically spindle-shaped morphology. Flow cytometry analysis showed that hLB-MSCs are positive for CD13, CD29, CD90, CD105, and CD106, but negative for CD3, CD4, CD5, CD11b, CD14, CD15, CD34, CD45, CD45RA, and HLA-DR. The detection of cell cycle from different passages indicated that hLB-MSCs have a similar potential for propagation during long culture in vitro. The most novel finding here is that, in addition to their mesodermal differentiation (osteoblasts and adipocytes), hLB-MSCs can also differentiated into extramesenchymal lineages, such as neural (ectoderm) and hepatic (endoderm) progenies. These results indicate that hLB-MSCs have a high level of plasticity and can differentiate into cell lineages from all three embryonic layers in vitro.

  11. Spatially defined InsP3-mediated signaling in embryonic stem cell-derived cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Nidhi Kapoor

    Full Text Available The functional role of inositol 1,4,5-trisphosphate (InsP3 signaling in cardiomyocytes is not entirely understood but it was linked to an increased propensity for triggered activity. The aim of this study was to determine how InsP3 receptors can translate Ca(2+ release into a depolarization of the plasma membrane and consequently arrhythmic activity. We used embryonic stem cell-derived cardiomyocytes (ESdCs as a model system since their spontaneous electrical activity depends on InsP3-mediated Ca(2+ release. [InsP3]i was monitored with the FRET-based InsP3-biosensor FIRE-1 (Fluorescent InsP3 Responsive Element and heterogeneity in sub-cellular [InsP3]i was achieved by targeted expression of FIRE-1 in the nucleus (FIRE-1nuc or expression of InsP3 5-phosphatase (m43 localized to the plasma membrane. Spontaneous activity of ESdCs was monitored simultaneously as cytosolic Ca(2+ transients (Fluo-4/AM and action potentials (current clamp. During diastole, the diastolic depolarization was paralleled by an increase of [Ca(2+]i and spontaneous activity was modulated by [InsP3]i. A 3.7% and 1.7% increase of FIRE-1 FRET ratio and 3.0 and 1.5 fold increase in beating frequency was recorded upon stimulation with endothelin-1 (ET-1, 100 nmol/L or phenylephrine (PE, 10 µmol/L, respectively. Buffering of InsP3 by FIRE-1nuc had no effect on the basal frequency while attenuation of InsP3 signaling throughout the cell (FIRE-1, or at the plasma membrane (m43 resulted in a 53.7% and 54.0% decrease in beating frequency. In m43 expressing cells the response to ET-1 was completely suppressed. Ca(2+ released from InsP3Rs is more effective than Ca(2+ released from RyRs to enhance INCX. The results support the hypothesis that in ESdCs InsP3Rs form a functional signaling domain with NCX that translates Ca(2+ release efficiently into a depolarization of the membrane potential.

  12. Evaluation of nefazodone-induced cardiotoxicity in human induced pluripotent stem cell-derived cardiomyocytes

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Sujeong, E-mail: crystalee@gmail.com [Next-generation Pharmaceutical Research Center, Korea Institute of Toxicology, Korea Research Institute of Chemical Technology, 141 Gajeong-ro, Yuseong-gu, Daejeon 305-343 (Korea, Republic of); Lee, Hyang-Ae, E-mail: hyangaelee@gmail.com [Next-generation Pharmaceutical Research Center, Korea Institute of Toxicology, Korea Research Institute of Chemical Technology, 141 Gajeong-ro, Yuseong-gu, Daejeon 305-343 (Korea, Republic of); Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, 110-799 (Korea, Republic of); Human and Environmental Toxicology Program, University of Science and Technology, 217 Gajeong-ro, Yuseong-gu, Daejeon 305-350 (Korea, Republic of); Choi, Sung Woo, E-mail: djmaya@snu.ac.kr [Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, 110-799 (Korea, Republic of); Kim, Sung Joon, E-mail: sjoonkim@snu.ac.kr [Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, 110-799 (Korea, Republic of); Kim, Ki-Suk, E-mail: idkks00@gmail.com [Next-generation Pharmaceutical Research Center, Korea Institute of Toxicology, Korea Research Institute of Chemical Technology, 141 Gajeong-ro, Yuseong-gu, Daejeon 305-343 (Korea, Republic of); Human and Environmental Toxicology Program, University of Science and Technology, 217 Gajeong-ro, Yuseong-gu, Daejeon 305-350 (Korea, Republic of)

    2016-04-01

    The recent establishment of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), which express the major cardiac ion channels and recapitulate spontaneous mechanical and electrical activities, may provide a possible solution for the lack of in vitro human-based cardiotoxicity testing models. Cardiotoxicity induced by the antidepressant nefazodone was previously revealed to cause an acquired QT prolongation by hERG channel blockade. To elucidate the cellular mechanisms underlying the cardiotoxicity of nefazodone beyond hERG, its effects on cardiac action potentials (APs) and ion channels were investigated using hiPSC-CMs with whole-cell patch clamp techniques. In a proof of principle study, we examined the effects of cardioactive channel blockers on the electrophysiological profile of hiPSC-CMs in advance of the evaluation of nefazodone. Nefazodone dose-dependently prolonged the AP duration at 90% (APD{sub 90}) and 50% (APD{sub 50}) repolarization, reduced the maximum upstroke velocity (dV/dt{sub max}) and induced early after depolarizations. Voltage-clamp studies of hiPSC-CMs revealed that nefazodone inhibited various voltage-gated ion channel currents including I{sub Kr}, I{sub Ks}, I{sub Na}, and I{sub Ca}. Among them, I{sub Kr} and I{sub Na} showed relatively higher sensitivity to nefazodone, consistent with the changes in the AP parameters. In summary, hiPSC-CMs enabled an integrated approach to evaluate the complex interactions of nefazodone with cardiac ion channels. These results suggest that hiPSC-CMs can be an effective model for detecting drug-induced arrhythmogenicity beyond the current standard assay of heterologously expressed hERG K{sup +} channels. - Highlights: • Nefazodone prolonged APD and decreased upstroke velocity of APs in hiPSC-CMs. • Nefazodone inhibited cardiac ion channels, especially I{sub Kr} and I{sub Na}, in hiPSC-CMs. • Nefazodone-induced AP changes are mainly the result of I{sub Kr} and I{sub Na} inhibition

  13. Evaluation of Optogenetic Electrophysiology Tools in Human Stem Cell-Derived Cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Susann Björk

    2017-11-01

    Full Text Available Current cardiac drug safety assessments focus on hERG channel block and QT prolongation for evaluating arrhythmic risks, whereas the optogenetic approach focuses on the action potential (AP waveform generated by a monolayer of human cardiomyocytes beating synchronously, thus assessing the contribution of several ion channels on the overall drug effect. This novel tool provides arrhythmogenic sensitizing by light-induced pacing in combination with non-invasive, all-optical measurements of cardiomyocyte APs and will improve assessment of drug-induced electrophysiological aberrancies. With the help of patch clamp electrophysiology measurements, we aimed to investigate whether the optogenetic modifications alter human cardiomyocytes' electrophysiology and how well the optogenetic analyses perform against this gold standard. Patch clamp electrophysiology measurements of non-transduced stem cell-derived cardiomyocytes compared to cells expressing the commercially available optogenetic constructs Optopatch and CaViar revealed no significant changes in action potential duration (APD parameters. Thus, inserting the optogenetic constructs into cardiomyocytes does not significantly affect the cardiomyocyte's electrophysiological properties. When comparing the two methods against each other (patch clamp vs. optogenetic imaging we found no significant differences in APD parameters for the Optopatch transduced cells, whereas the CaViar transduced cells exhibited modest increases in APD-values measured with optogenetic imaging. Thus, to broaden the screen, we combined optogenetic measurements of membrane potential and calcium transients with contractile motion measured by video motion tracking. Furthermore, to assess how optogenetic measurements can predict changes in membrane potential, or early afterdepolarizations (EADs, cells were exposed to cumulating doses of E-4031, a hERG potassium channel blocker, and drug effects were measured at both spontaneous and

  14. Lethal and mutagenic effects of radiation and chemicals on cultured fish cells derived the erythrophoroma of goldfish (Carassius auratus)

    Energy Technology Data Exchange (ETDEWEB)

    Mitani, H. (Tokyo Univ. (Japan). Inst. of Zoology)

    1983-01-01

    GEM 199 cells derived from an eryhtrophoroma of goldfish (Carassius auratus), which had a high plating efficiency, were used to investigate the lethal and mutational effects of radiations (UV and ..gamma..-rays) and chemicals (4NQO and MNNG). The cells were more resistant to rays than mammalian cells and CAF-MM1 cells derived from the normal fin tissue of goldfish. They were also more resistant to UV-irradiation than CAF-MM1 cells. Photoreactivation after UV-irradiation was present in GEM 199 cells for both survival and mutation. The initial shoulder of the survival curve of UV-irradiated cells was reduced greatly by caffeine, suggesting a high activity of the post-replication repair. The spontaneous mutation frequency to ouabain resistance was 1-5x10/sup -6/ clones per viable cell. MNNG was effective in inducing ouabain-resistant mutation, while 4NQO and ..gamma..-rays did not induce mutation.

  15. High-Content Electrophysiological Analysis of Human Pluripotent Stem Cell-Derived Cardiomyocytes (hPSC-CMs).

    Science.gov (United States)

    Kong, Chi-Wing; Geng, Lin; Li, Ronald A

    2018-01-01

    Considerable interest has been raised to develop human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) as a model for drug discovery and cardiotoxicity screening. High-content electrophysiological analysis of currents generated by transmembrane cell surface ion channels has been pursued to complement such emerging applications. Here we describe practical procedures and considerations for accomplishing successful assays of hPSC-CMs using an automated planar patch-clamp system.

  16. Transplantation of human embryonic stem cell-derived oligodendrocyte progenitors into rat spinal cord injuries does not cause harm.

    Science.gov (United States)

    Cloutier, Frank; Siegenthaler, Monica M; Nistor, Gabriel; Keirstead, Hans S

    2006-07-01

    Demyelination contributes to loss of function following spinal cord injury. We have shown previously that transplantation of human embryonic stem cell-derived oligodendrocyte progenitors into adult rat 200 kD contusive spinal cord injury sites enhances remyelination and promotes recovery of motor function. Previous studies using oligodendrocyte lineage cells have noted a correlation between the presence of demyelinating pathology and the survival and migration rate of the transplanted cells. The present study compared the survival and migration of human embryonic stem cell-derived oligodendrocyte progenitors injected 7 days after a 200 or 50 kD contusive spinal cord injury, as well as the locomotor outcome of transplantation. Our findings indicate that a 200 kD spinal cord injury induces extensive demyelination, whereas a 50 kD spinal cord injury induces no detectable demyelination. Cells transplanted into the 200 kD injury group survived, migrated, and resulted in robust remyelination, replicating our previous studies. In contrast, cells transplanted into the 50 kD injury group survived, exhibited limited migration, and failed to induce remyelination as demyelination in this injury group was absent. Animals that received a 50 kD injury displayed only a transient decline in locomotor function as a result of the injury. Importantly, human embryonic stem cell-derived oligodendrocyte progenitor transplants into the 50 kD injury group did not cause a further decline in locomotion. Our studies highlight the importance of a demyelinating pathology as a prerequisite for the function of transplanted myelinogenic cells. In addition, our results indicate that transplantation of human embryonic stem cell-derived oligodendrocyte progenitor cells into the injured spinal cord is not associated with a decline in locomotor function.

  17. Transplant of stem cells derived from bone marrow and granulocytic growth factor in acute and chronic ischemic myocardiopathy

    International Nuclear Information System (INIS)

    Senior Juan M; Cuellar Francisco; Velasquez Oscar; Velasquez Margarita; Navas Claudia M; Ortiz Sergio; Delgado Juan A; Guillerrno, Blanco; Londono Juan L; Coronado Manuel A; Gomez Francisco; Alzate, Fernando Leon; Zuluaga Alejandra

    2007-01-01

    Recent studies have shown the safety and efficacy of the stem cells derived from bone marrow (BMC) implant with concomitant administration of stimulating factor of granulocyte colonies in patients with acute myocardial infarction with ST segment elevation and in chronic ischemic cardiopathy. An open prospective (before and after) design was made to evaluate the safety and efficacy of cell therapy associated to growth factor administration. The first experience with this kind of therapy is reported. Methodology: this is a 6 months follow-up report of patients with acute and chronic ischemic cardiopathy to who transplant of stem cells derived from bone marrow mobilized with granulocyte colonies growth stimulating factor via coronary arteries or epicardium was realized. Two groups of patients were included: Ten patients with anterior wall infarct and 2. Five patients with chronic ischemic cardiopathy, all with extensive necrosis demonstrated by absence of myocardial viability through nuclear medicine and ejection fraction of less than 40%. Results: significant improvement of ejection fraction from 29.44 ± 3.36 to 37.6 ± 5.3 with p<0.001 and decrease of ventricular systolic and diastolic volume without statistical significance (p =0.31 and 0.4 respectively) were demonstrated. Exercise capacity evidenced by increment in the six minutes test, exercise time and the MET number achieved, increased in a significant way. There were significant changes in the perfusion defect from the second follow-up month and no complications directly related to the stem cells derived from bone marrow transplant or the use of stimulating granulocyte colony factor were presented. Conclusions: this is the first experience of stem cells derived from bone marrow transplant associated to the administration of stimulating granulocyte growth colony factor in which recovery of left ventricular function was demonstrated, as well as improvement in exercise capacity and in the perfusion defect

  18. Human Induced Pluripotent Stem Cell-Derived Cardiac Progenitor Cells in Phenotypic Screening: A Transforming Growth Factor-β Type 1 Receptor Kinase Inhibitor Induces Efficient Cardiac Differentiation.

    Science.gov (United States)

    Drowley, Lauren; Koonce, Chad; Peel, Samantha; Jonebring, Anna; Plowright, Alleyn T; Kattman, Steven J; Andersson, Henrik; Anson, Blake; Swanson, Bradley J; Wang, Qing-Dong; Brolen, Gabriella

    2016-02-01

    Several progenitor cell populations have been reported to exist in hearts that play a role in cardiac turnover and/or repair. Despite the presence of cardiac stem and progenitor cells within the myocardium, functional repair of the heart after injury is inadequate. Identification of the signaling pathways involved in the expansion and differentiation of cardiac progenitor cells (CPCs) will broaden insight into the fundamental mechanisms playing a role in cardiac homeostasis and disease and might provide strategies for in vivo regenerative therapies. To understand and exploit cardiac ontogeny for drug discovery efforts, we developed an in vitro human induced pluripotent stem cell-derived CPC model system using a highly enriched population of KDR(pos)/CKIT(neg)/NKX2.5(pos) CPCs. Using this model system, these CPCs were capable of generating highly enriched cultures of cardiomyocytes under directed differentiation conditions. In order to facilitate the identification of pathways and targets involved in proliferation and differentiation of resident CPCs, we developed phenotypic screening assays. Screening paradigms for therapeutic applications require a robust, scalable, and consistent methodology. In the present study, we have demonstrated the suitability of these cells for medium to high-throughput screens to assess both proliferation and multilineage differentiation. Using this CPC model system and a small directed compound set, we identified activin-like kinase 5 (transforming growth factor-β type 1 receptor kinase) inhibitors as novel and potent inducers of human CPC differentiation to cardiomyocytes. Significance: Cardiac disease is a leading cause of morbidity and mortality, with no treatment available that can result in functional repair. This study demonstrates how differentiation of induced pluripotent stem cells can be used to identify and isolate cell populations of interest that can translate to the adult human heart. Two separate examples of phenotypic

  19. Nano-zymography Using Laser-Scanning Confocal Microscopy Unmasks Proteolytic Activity of Cell-Derived Microparticles.

    Science.gov (United States)

    Briens, Aurélien; Gauberti, Maxime; Parcq, Jérôme; Montaner, Joan; Vivien, Denis; Martinez de Lizarrondo, Sara

    2016-01-01

    Cell-derived microparticles (MPs) are nano-sized vesicles released by activated cells in the extracellular milieu. They act as vectors of biological activity by carrying membrane-anchored and cytoplasmic constituents of the parental cells. Although detection and characterization of cell-derived MPs may be of high diagnostic and prognostic values in a number of human diseases, reliable measurement of their size, number and biological activity still remains challenging using currently available methods. In the present study, we developed a protocol to directly image and functionally characterize MPs using high-resolution laser-scanning confocal microscopy. Once trapped on annexin-V coated micro-wells, we developed several assays using fluorescent reporters to measure their size, detect membrane antigens and evaluate proteolytic activity (nano-zymography). In particular, we demonstrated the applicability and specificity of this method to detect antigens and proteolytic activities of tissue-type plasminogen activator (tPA), urokinase and plasmin at the surface of engineered MPs from transfected cell-lines. Furthermore, we were able to identify a subset of tPA-bearing fibrinolytic MPs using plasma samples from a cohort of ischemic stroke patients who received thrombolytic therapy and in an experimental model of thrombin-induced ischemic stroke in mice. Overall, this method is promising for functional characterization of cell-derived MPs.

  20. Cell-Derived Microparticles in Patients with Type 2 Diabetes Mellitus: a Systematic Review and Meta-Analysis

    Directory of Open Access Journals (Sweden)

    Sheyu Li

    2016-11-01

    Full Text Available Background/Aims: The aim of this study was to assess the association between circulating cell-derived microparticles (MPs and type 2 diabetes mellitus (T2DM. Methods: A literature search was performed systematically in PubMed and Embase to identify available case-control or cross-sectional studies that compared different types of cell-derived MPs in patients with T2DM and non-diabetic controls. Pooled standardized mean differences (SMDs of each MP type were pooled using meta-analysis. Results: Forty-eight studies involving 2,460 patients with T2DM and 1,880 non-diabetic controls were included for systematic review and 34 of which were included for quantitative study by meta-analysis. In the overall analysis, the levels of circulating total MPs (TMPs, platelet-derived MPs (PMPs, monocyte-derived MPs (MMPs and endothelium-derived MPs (EMPs were significantly higher in T2DM patients than those in controls (TMPs: SMD, 0.64; 95%CI, 0.12∼1.15; P=0.02; PMPs: SMD, 1.19; 95%CI, 0.88∼1.50; P Conclusions: The counts of TMPs, PMPs, MMPs and EMPs elevated in patients with T2DM. And cell-derived MPs may play a role in the pathogenesis of T2DM.

  1. Molecular and in vivo characterization of cancer-propagating cells derived from MYCN-dependent medulloblastoma.

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    Zai Ahmad

    Full Text Available Medulloblastoma (MB is the most common malignant pediatric brain tumor. While the pathways that are deregulated in MB remain to be fully characterized, amplification and/or overexpression of the MYCN gene, which is has a critical role in cerebellar development as a regulator of neural progenitor cell fate, has been identified in several MB subgroups. Phenotypically, aberrant expression of MYCN is associated with the large-cell/anaplastic MB variant, which accounts for 5-15% of cases and is associated with aggressive disease and poor clinical outcome. To better understand the role of MYCN in MB in vitro and in vivo and to aid the development of MYCN-targeted therapeutics we established tumor-derived neurosphere cell lines from the GTML (Glt1-tTA/TRE-MYCN-Luc genetically engineered mouse model. A fraction of GTML neurospheres were found to be growth factor independent, expressed CD133 (a marker of neural stem cells, failed to differentiate upon MYCN withdrawal and were highly tumorigenic when orthotopically implanted into the cerebellum. Principal component analyzes using single cell RNA assay data suggested that the clinical candidate aurora-A kinase inhibitor MLN8237 converts GTML neurospheres to resemble non-MYCN expressors. Correlating with this, MLN8237 significantly extended the survival of mice bearing GTML MB allografts. In summary, our results demonstrate that MYCN plays a critical role in expansion and survival of aggressive MB-propagating cells, and establish GTML neurospheres as an important resource for the development of novel therapeutic strategies.

  2. Retinal vascular injuries and intravitreal human embryonic stem cell-derived haemangioblasts.

    Science.gov (United States)

    Wang, Jin-Da; An, Ying; Zhang, Jing-Shang; Wan, Xiu-Hua; Zhang, Wei; Lanza, Robert; Lu, Shi-Jiang; Jonas, Jost B; Xu, Liang

    2017-09-01

    To investigate whether intravitreally applied haemangioblasts (HB) derived from human embryonic stem cells (hESCs) are helpful for the repair of vascular damage caused in animals by an oxygen-induced retinopathy (OIR), by an induced diabetic retinopathy (DR) or by an induced retinal ischaemia with subsequent reperfusion. Human embryonic stem cell-derived HBs were transplanted intravitreally into C57BL/6J mice (OIR model), into male Wistar rats with an induced DR and into male Wistar rats undergoing induced retinal ischaemia with subsequent reperfusion. Control groups of animals received an intravitreal injection of endothelial cells (ECs) or phosphate-buffered saline (PBS). We examined the vasculature integrity in the mice with OIR, the blood-retina barrier in the rats with induced DR, and retinal thickness and retinal ganglion cell density in retina flat mounts of the rats with the retinal ischaemic-reperfusion retinopathy. In the OIR model, the study group versus control groups showed a significantly (p < 0.001) smaller retinal avascular area [5.1 ± 2.7%;n = 18 animals versus 12.2 ± 2.8% (PBS group; n = 10 animals) and versus 11.8 ± 3.7% (EC group; n = 8 animals)] and less retinal neovascularization [6.3 ± 2.5%;n = 18 versus 15.2 ± 6.3% (n = 10; PBS group) and versus 15.8 ± 3.3% (n = 8; EC group)]. On retinal flat mounts, hESC-HBs were integrated into damaged retinal vessels and stained positive for PECAM (CD31) as EC marker. In the DR model, the study group versus the EC control group showed a significantly (p = 0.001) better blood-retina barrier function as measured at 2 days after the intravitreal injections [study group: 20.2 ± 12.8 μl/(g × hr); n = 6; versus EC control group: 52.9 ± 9.9 μl/(g × hr; n = 6)]. In the retinal ischaemia-reperfusion model, the groups did not differ significantly in retinal thickness and retinal ganglion cell density at 2, 5 and 7 days after baseline. By integrating into

  3. Temporal and Spatial Simulation of Atmospheric Pollutant PM2.5 Changes and Risk Assessment of Population Exposure to Pollution Using Optimization Algorithms of the Back Propagation-Artificial Neural Network Model and GIS

    Directory of Open Access Journals (Sweden)

    Ping Zhang

    2015-09-01

    Full Text Available PM2.5 pollution has become of increasing public concern because of its relative importance and sensitivity to population health risks. Accurate predictions of PM2.5 pollution and population exposure risks are crucial to developing effective air pollution control strategies. We simulated and predicted the temporal and spatial changes of PM2.5 concentration and population exposure risks, by coupling optimization algorithms of the Back Propagation-Artificial Neural Network (BP-ANN model and a geographical information system (GIS in Xi’an, China, for 2013, 2020, and 2025. Results indicated that PM2.5 concentration was positively correlated with GDP, SO2, and NO2, while it was negatively correlated with population density, average temperature, precipitation, and wind speed. Principal component analysis of the PM2.5 concentration and its influencing factors’ variables extracted four components that accounted for 86.39% of the total variance. Correlation coefficients of the Levenberg-Marquardt (trainlm and elastic (trainrp algorithms were more than 0.8, the index of agreement (IA ranged from 0.541 to 0.863 and from 0.502 to 0.803 by trainrp and trainlm algorithms, respectively; mean bias error (MBE and Root Mean Square Error (RMSE indicated that the predicted values were very close to the observed values, and the accuracy of trainlm algorithm was better than the trainrp. Compared to 2013, temporal and spatial variation of PM2.5 concentration and risk of population exposure to pollution decreased in 2020 and 2025. The high-risk areas of population exposure to PM2.5 were mainly distributed in the northern region, where there is downtown traffic, abundant commercial activity, and more exhaust emissions. A moderate risk zone was located in the southern region associated with some industrial pollution sources, and there were mainly low-risk areas in the western and eastern regions, which are predominantly residential and educational areas.

  4. Temporal and Spatial Simulation of Atmospheric Pollutant PM2.5 Changes and Risk Assessment of Population Exposure to Pollution Using Optimization Algorithms of the Back Propagation-Artificial Neural Network Model and GIS.

    Science.gov (United States)

    Zhang, Ping; Hong, Bo; He, Liang; Cheng, Fei; Zhao, Peng; Wei, Cailiang; Liu, Yunhui

    2015-09-29

    PM2.5 pollution has become of increasing public concern because of its relative importance and sensitivity to population health risks. Accurate predictions of PM2.5 pollution and population exposure risks are crucial to developing effective air pollution control strategies. We simulated and predicted the temporal and spatial changes of PM2.5 concentration and population exposure risks, by coupling optimization algorithms of the Back Propagation-Artificial Neural Network (BP-ANN) model and a geographical information system (GIS) in Xi'an, China, for 2013, 2020, and 2025. Results indicated that PM2.5 concentration was positively correlated with GDP, SO₂, and NO₂, while it was negatively correlated with population density, average temperature, precipitation, and wind speed. Principal component analysis of the PM2.5 concentration and its influencing factors' variables extracted four components that accounted for 86.39% of the total variance. Correlation coefficients of the Levenberg-Marquardt (trainlm) and elastic (trainrp) algorithms were more than 0.8, the index of agreement (IA) ranged from 0.541 to 0.863 and from 0.502 to 0.803 by trainrp and trainlm algorithms, respectively; mean bias error (MBE) and Root Mean Square Error (RMSE) indicated that the predicted values were very close to the observed values, and the accuracy of trainlm algorithm was better than the trainrp. Compared to 2013, temporal and spatial variation of PM2.5 concentration and risk of population exposure to pollution decreased in 2020 and 2025. The high-risk areas of population exposure to PM2.5 were mainly distributed in the northern region, where there is downtown traffic, abundant commercial activity, and more exhaust emissions. A moderate risk zone was located in the southern region associated with some industrial pollution sources, and there were mainly low-risk areas in the western and eastern regions, which are predominantly residential and educational areas.

  5. Neural Networks

    Directory of Open Access Journals (Sweden)

    Schwindling Jerome

    2010-04-01

    Full Text Available This course presents an overview of the concepts of the neural networks and their aplication in the framework of High energy physics analyses. After a brief introduction on the concept of neural networks, the concept is explained in the frame of neuro-biology, introducing the concept of multi-layer perceptron, learning and their use as data classifer. The concept is then presented in a second part using in more details the mathematical approach focussing on typical use cases faced in particle physics. Finally, the last part presents the best way to use such statistical tools in view of event classifers, putting the emphasis on the setup of the multi-layer perceptron. The full article (15 p. corresponding to this lecture is written in french and is provided in the proceedings of the book SOS 2008.

  6. Ketamine Causes Mitochondrial Dysfunction in Human Induced Pluripotent Stem Cell-Derived Neurons

    Science.gov (United States)

    Ito, Hiroyuki; Uchida, Tokujiro; Makita, Koshi

    2015-01-01

    Purpose Ketamine toxicity has been demonstrated in nonhuman mammalian neurons. To study the toxic effect of ketamine on human neurons, an experimental model of cultured neurons from human induced pluripotent stem cells (iPSCs) was examined, and the mechanism of its toxicity was investigated. Methods Human iPSC-derived dopaminergic neurons were treated with 0, 20, 100 or 500 μM ketamine for 6 and 24 h. Ketamine toxicity was evaluated by quantification of caspase 3/7 activity, reactive oxygen species (ROS) production, mitochondrial membrane potential, ATP concentration, neurotransmitter reuptake activity and NADH/NAD+ ratio. Mitochondrial morphological change was analyzed by transmission electron microscopy and confocal microscopy. Results Twenty-four-hour exposure of iPSC-derived neurons to 500 μM ketamine resulted in a 40% increase in caspase 3/7 activity (P ketamine (100 μM) decreased the ATP level (22%, P ketamine concentration, which suggests that mitochondrial dysfunction preceded ROS generation and caspase activation. Conclusions We established an in vitro model for assessing the neurotoxicity of ketamine in iPSC-derived neurons. The present data indicate that the initial mitochondrial dysfunction and autophagy may be related to its inhibitory effect on the mitochondrial electron transport system, which underlies ketamine-induced neural toxicity. Higher ketamine concentration can induce ROS generation and apoptosis in human neurons. PMID:26020236

  7. Microparticle Shedding from Neural Progenitor Cells and Vascular Compartment Cells Is Increased in Ischemic Stroke.

    Science.gov (United States)

    Chiva-Blanch, Gemma; Suades, Rosa; Crespo, Javier; Peña, Esther; Padró, Teresa; Jiménez-Xarrié, Elena; Martí-Fàbregas, Joan; Badimon, Lina

    2016-01-01

    Ischemic stroke has shown to induce platelet and endothelial microparticle shedding, but whether stroke induces microparticle shedding from additional blood and vascular compartment cells is unclear. Neural precursor cells have been shown to replace dying neurons at sites of brain injury; however, if neural precursor cell activation is associated to microparticle shedding, and whether this activation is maintained at long term and associates to stroke type and severity remains unknown. We analyzed neural precursor cells and blood and vascular compartment cells microparticle shedding after an acute ischemic stroke. Forty-four patients were included in the study within the first 48h after the onset of stroke. The cerebral lesion size was evaluated at 3-7 days of the stroke. Circulating microparticles from neural precursor cells and blood and vascular compartment cells (platelets, endothelial cells, erythrocytes, leukocytes, lymphocytes, monocytes and smooth muscle cells) were analyzed by flow cytometry at the onset of stroke and at 7 and 90 days. Forty-four age-matched high cardiovascular risk subjects without documented vascular disease were used as controls. Compared to high cardiovascular risk controls, patients showed higher number of neural precursor cell- and all blood and vascular compartment cell-derived microparticles at the onset of stroke, and after 7 and 90 days. At 90 days, neural precursor cell-derived microparticles decreased and smooth muscle cell-derived microparticles increased compared to levels at the onset of stroke, but only in those patients with the highest stroke-induced cerebral lesions. Stroke increases blood and vascular compartment cell and neural precursor cell microparticle shedding, an effect that is chronically maintained up to 90 days after the ischemic event. These results show that stroke induces a generalized blood and vascular cell activation and the initiation of neuronal cell repair process after stroke. Larger cerebral lesions

  8. Microparticle Shedding from Neural Progenitor Cells and Vascular Compartment Cells Is Increased in Ischemic Stroke.

    Directory of Open Access Journals (Sweden)

    Gemma Chiva-Blanch

    Full Text Available Ischemic stroke has shown to induce platelet and endothelial microparticle shedding, but whether stroke induces microparticle shedding from additional blood and vascular compartment cells is unclear. Neural precursor cells have been shown to replace dying neurons at sites of brain injury; however, if neural precursor cell activation is associated to microparticle shedding, and whether this activation is maintained at long term and associates to stroke type and severity remains unknown. We analyzed neural precursor cells and blood and vascular compartment cells microparticle shedding after an acute ischemic stroke.Forty-four patients were included in the study within the first 48h after the onset of stroke. The cerebral lesion size was evaluated at 3-7 days of the stroke. Circulating microparticles from neural precursor cells and blood and vascular compartment cells (platelets, endothelial cells, erythrocytes, leukocytes, lymphocytes, monocytes and smooth muscle cells were analyzed by flow cytometry at the onset of stroke and at 7 and 90 days. Forty-four age-matched high cardiovascular risk subjects without documented vascular disease were used as controls.Compared to high cardiovascular risk controls, patients showed higher number of neural precursor cell- and all blood and vascular compartment cell-derived microparticles at the onset of stroke, and after 7 and 90 days. At 90 days, neural precursor cell-derived microparticles decreased and smooth muscle cell-derived microparticles increased compared to levels at the onset of stroke, but only in those patients with the highest stroke-induced cerebral lesions.Stroke increases blood and vascular compartment cell and neural precursor cell microparticle shedding, an effect that is chronically maintained up to 90 days after the ischemic event. These results show that stroke induces a generalized blood and vascular cell activation and the initiation of neuronal cell repair process after stroke. Larger

  9. Stem cell-derived vascular endothelial cells and their potential application in regenerative medicine

    Science.gov (United States)

    Although a 'vascular stem cell' population has not been identified or generated, vascular endothelial and mural cells (smooth muscle cells and pericytes) can be derived from currently known pluripotent stem cell sources, including human embryonic stem cells and induced pluripotent stem cells. We rev...

  10. Completely ES cell-derived mice produced by tetraploid complementation using inner cell mass (ICM deficient blastocysts.

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    Duancheng Wen

    Full Text Available Tetraploid complementation is often used to produce mice from embryonic stem cells (ESCs by injection of diploid (2n ESCs into tetraploid (4n blastocysts (ESC-derived mice. This method has also been adapted to mouse cloning and the derivation of mice from induced pluripotent stem (iPS cells. However, the underlying mechanism(s of the tetraploid complementation remains largely unclear. Whether this approach can give rise to completely ES cell-derived mice is an open question, and has not yet been unambiguously proven. Here, we show that mouse tetraploid blastocysts can be classified into two groups, according to the presence or absence of an inner cell mass (ICM. We designate these as type a (presence of ICM at blastocyst stage or type b (absence of ICM. ESC lines were readily derived from type a blastocysts, suggesting that these embryos retain a pluripotent epiblast compartment; whereas the type b blastocysts possessed very low potential to give rise to ESC lines, suggesting that they had lost the pluripotent epiblast. When the type a blastocysts were used for tetraploid complementation, some of the resulting mice were found to be 2n/4n chimeric; whereas when type b blastocysts were used as hosts, the resulting mice are all completely ES cell-derived, with the newborn pups displaying a high frequency of abdominal hernias. Our results demonstrate that completely ES cell-derived mice can be produced using ICM-deficient 4n blastocysts, and provide evidence that the exclusion of tetraploid cells from the fetus in 2n/4n chimeras can largely be attributed to the formation of ICM-deficient blastocysts.

  11. Cell-Derived Microparticles in Patients with Type 2 Diabetes Mellitus: a Systematic Review and Meta-Analysis.

    Science.gov (United States)

    Li, Sheyu; Wei, Jia; Zhang, Chenghui; Li, Xiaodan; Meng, Wentong; Mo, Xianming; Zhang, Qianying; Liu, Qilin; Ren, Kaiyun; Du, Rong; Tian, Haoming; Li, Jianwei

    2016-01-01

    The aim of this study was to assess the association between circulating cell-derived microparticles (MPs) and type 2 diabetes mellitus (T2DM). A literature search was performed systematically in PubMed and Embase to identify available case-control or cross-sectional studies that compared different types of cell-derived MPs in patients with T2DM and non-diabetic controls. Pooled standardized mean differences (SMDs) of each MP type were pooled using meta-analysis. Forty-eight studies involving 2,460 patients with T2DM and 1,880 non-diabetic controls were included for systematic review and 34 of which were included for quantitative study by meta-analysis. In the overall analysis, the levels of circulating total MPs (TMPs), platelet-derived MPs (PMPs), monocyte-derived MPs (MMPs) and endothelium-derived MPs (EMPs) were significantly higher in T2DM patients than those in controls (TMPs: SMD, 0.64; 95%CI, 0.12∼1.15; P=0.02; PMPs: SMD, 1.19; 95%CI, 0.88∼1.50; P <0.00001; MMPs: SMD, 0.92; 95%CI, 0.66∼1.17; P <0.00001; EMPs: SMD, 0.73; 95%CI, 0.50∼0.96; P <0.00001). Meanwhile, no significant difference was shown in leukocyte-derived MPs (LMPs) level between diabetic and non-diabetic groups (SMD, 0.37; 95%CI, -0.15∼0.89; P=0.17). The counts of TMPs, PMPs, MMPs and EMPs elevated in patients with T2DM. And cell-derived MPs may play a role in the pathogenesis of T2DM. © 2016 The Author(s) Published by S. Karger AG, Basel.

  12. Collaborative Recurrent Neural Networks forDynamic Recommender Systems

    Science.gov (United States)

    2016-11-22

    JMLR: Workshop and Conference Proceedings 63:366–381, 2016 ACML 2016 Collaborative Recurrent Neural Networks for Dynamic Recommender Systems Young...an unprece- dented scale. Although such activity logs are abundantly available, most approaches to recommender systems are based on the rating...Recurrent Neural Network, Recommender System , Neural Language Model, Collaborative Filtering 1. Introduction As ever larger parts of the population

  13. Human-induced pluripotent stem cell-derived cardiomyocytes from cardiac progenitor cells: effects of selective ion channel blockade.

    Science.gov (United States)

    Altomare, Claudia; Pianezzi, Enea; Cervio, Elisabetta; Bolis, Sara; Biemmi, Vanessa; Benzoni, Patrizia; Camici, Giovanni G; Moccetti, Tiziano; Barile, Lucio; Vassalli, Giuseppe

    2016-12-01

    Human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are likely to revolutionize electrophysiological approaches to arrhythmias. Recent evidence suggests the somatic cell origin of hiPSCs may influence their differentiation potential. Owing to their cardiomyogenic potential, cardiac-stromal progenitor cells (CPCs) are an interesting cellular source for generation of hiPSC-derived cardiomyocytes. The effect of ionic current blockade in hiPSC-derived cardiomyocytes generated from CPCs has not been characterized yet. Human-induced pluripotent stem cell-derived cardiomyocytes were generated from adult CPCs and skin fibroblasts from the same individuals. The effect of selective ionic current blockade on spontaneously beating hiPSC-derived cardiomyocytes was assessed using multi-electrode arrays. Cardiac-stromal progenitor cells could be reprogrammed into hiPSCs, then differentiated into hiPSC-derived cardiomyocytes. Human-induced pluripotent stem cell-derived cardiomyocytes of cardiac origin showed higher upregulation of cardiac-specific genes compared with those of fibroblastic origin. Human-induced pluripotent stem cell-derived cardiomyocytes of both somatic cell origins exhibited sensitivity to tetrodotoxin, a blocker of Na +  current (I Na ), nifedipine, a blocker of L-type Ca 2+  current (I CaL ), and E4031, a blocker of the rapid component of delayed rectifier K +  current (I Kr ). Human-induced pluripotent stem cell-derived cardiomyocytes of cardiac origin exhibited sensitivity to JNJ303, a blocker of the slow component of delayed rectifier K +  current (I Ks ). In hiPSC-derived cardiomyocytes of cardiac origin, I Na , I CaL , I Kr , and I Ks were present as tetrodotoxin-, nifedipine-, E4031-, and JNJ303-sensitive currents, respectively. Although cardiac differentiation efficiency was improved in hiPSCs of cardiac vs. non-cardiac origin, no major functional differences were observed between hiPSC-derived cardiomyocytes of different somatic

  14. SAFETY OF CELL-DERIVED SUBUNIT ADJUVANTED INFLUENZA VACCINE FOR CHILDREN VACCINATION: DOUBLE-BLIND RANDOMIZED CLINICAL TRIAL

    Directory of Open Access Journals (Sweden)

    S.M. Kharit

    2010-01-01

    Full Text Available This article presents the safety data for cell-derived inactivated subunit adjuvanted influenza vaccine «Grippol Neo» in children 3–17 years old in comparison with reference egg-derived inactivated subunit vaccine «Grippol plus». Good test vaccine tolerability and high efficacy profile is demonstrated. Based on the results obtained vaccine «Grippol Neo» is recommended for mass influenza prophylaxis in pediatry, including National Immunization Schedule.Key words: children, influenza, vaccination, «Grippol Neo».(Voprosy sovremennoi pediatrii — Current Pediatrics. – 2010;9(4:44-49

  15. Generation and characterization of human iPSC line generated from mesenchymal stem cells derived from adipose tissue.

    Science.gov (United States)

    Zapata-Linares, Natalia; Rodriguez, Saray; Mazo, Manuel; Abizanda, Gloria; Andreu, Enrique J; Barajas, Miguel; Prosper, Felipe; Rodriguez-Madoz, Juan R

    2016-01-01

    In this work, mesenchymal stem cells derived from adipose tissue (ADSCs) were used for the generation of the human-induced pluripotent stem cell line G15.AO. Cell reprogramming was performed using retroviral vectors containing the Yamanaka factors, and the generated G15.AO hiPSC line showed normal karyotype, silencing of the exogenous reprogramming factors, induction of the typical pluripotency-associated markers, alkaline phosphatase enzymatic activity, and in vivo and in vitro differentiation ability to the three germ layers. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  16. A transduced living hyaline cartilage graft releasing transgenic stromal cell-derived factor-1 inducing endogenous stem cell homing in vivo.

    Science.gov (United States)

    Zhang, Feng; Leong, Wenyan; Su, Kai; Fang, Yu; Wang, Dong-An

    2013-05-01

    Stromal cell-derived factor-1 (SDF-1), also known as a homing factor, is a potent chemokine that activates and directs mobilization, migration, and retention of certain cell species via systemic circulation. The responding homing cells largely consist of activated stem cells, so that, in case of tissue lesions, such SDF-1-induced cell migration may execute recruitment of endogenous stem cells to perform autoreparation and compensatory regeneration in situ. In this study, a recombinant adenoviral vector carrying SDF-1 transgene was constructed and applied to transduce a novel scaffold-free living hyaline cartilage graft (SDF-t-LhCG). As an engineered transgenic living tissue, SDF-t-LhCG is capable of continuously producing and releasing SDF-1 in vitro and in vivo. The in vitro trials were examined with ELISA, while the in vivo trials were subsequently performed via a subcutaneous implantation of SDF-t-LhCG in a nude mouse model, followed by series of biochemical and biological analyses. The results indicate that transgenic SDF-1 enhanced the presence of this chemokine in mouse's circulation system; in consequence, SDF-1-induced activation and recruitment of endogenous stem cells were also augmented in both peripheral blood and SDF-t-LhCG implant per se. These results were obtained via flow cytometry analyses on mouse blood samples and implanted SDF-t-LhCG samples, indicating an upregulation of the CXCR4(+)(SDF-1 receptor) cell population, accompanied by upregulation of the CD34(+), CD44(+), and Sca-1(+) cell populations as well as a downregulation of the CD11b(+) cell population. With the supply of SDF-1-recruited endogenous stem cells, enhanced chondrogenesis was observed in SDF-t-LhCG implants in situ.

  17. A population of serumdeprivation-induced bone marrow stem cells (SD-BMSC) expresses marker typical for embryonic and neural stem cells

    International Nuclear Information System (INIS)

    Sauerzweig, Steven; Munsch, Thomas; Lessmann, Volkmar; Reymann, Klaus G.; Braun, Holger

    2009-01-01

    The bone marrow represents an easy accessible source of adult stem cells suitable for various cell based therapies. Several studies in recent years suggested the existence of pluripotent stem cells within bone marrow stem cells (BMSC) expressing marker proteins of both embryonic and tissue committed stem cells. These subpopulations were referred to as MAPC, MIAMI and VSEL-cells. Here we describe SD-BMSC (serumdeprivation-induced BMSC) which are induced as a distinct subpopulation after complete serumdeprivation. SD-BMSC are generated from small-sized nestin-positive BMSC (S-BMSC) organized as round-shaped cells in the top layer of BMSC-cultures. The generation of SD-BMSC is caused by a selective proliferation of S-BMSC and accompanied by changes in both morphology and gene expression. SD-BMSC up-regulate not only markers typical for neural stem cells like nestin and GFAP, but also proteins characteristic for embryonic cells like Oct4 and SOX2. We hypothesize, that SD-BMSC like MAPC, MIAMI and VSEL-cells represent derivatives from a single pluripotent stem cell fraction within BMSC exhibiting characteristics of embryonic and tissue committed stem cells. The complete removal of serum might offer a simple way to specifically enrich this fraction of pluripotent embryonic like stem cells in BMSC cultures

  18. Increased IgG on cell-derived plasma microparticles in systemic lupus erythematosus is associated with autoantibodies and complement activation

    DEFF Research Database (Denmark)

    Nielsen, Christoffer T; Østergaard, Ole; Stener, Line

    2012-01-01

    To quantify immunoglobulin and C1q on circulating cell-derived microparticles (MPs) in patients with systemic lupus erythematosus (SLE) and to determine whether immunoglobulin and C1q levels are correlated with clinical and serologic parameters.......To quantify immunoglobulin and C1q on circulating cell-derived microparticles (MPs) in patients with systemic lupus erythematosus (SLE) and to determine whether immunoglobulin and C1q levels are correlated with clinical and serologic parameters....

  19. Neural crest contributions to the lamprey head

    Science.gov (United States)

    McCauley, David W.; Bronner-Fraser, Marianne

    2003-01-01

    The neural crest is a vertebrate-specific cell population that contributes to the facial skeleton and other derivatives. We have performed focal DiI injection into the cranial neural tube of the developing lamprey in order to follow the migratory pathways of discrete groups of cells from origin to destination and to compare neural crest migratory pathways in a basal vertebrate to those of gnathostomes. The results show that the general pathways of cranial neural crest migration are conserved throughout the vertebrates, with cells migrating in streams analogous to the mandibular and hyoid streams. Caudal branchial neural crest cells migrate ventrally as a sheet of cells from the hindbrain and super-pharyngeal region of the neural tube and form a cylinder surrounding a core of mesoderm in each pharyngeal arch, similar to that seen in zebrafish and axolotl. In addition to these similarities, we also uncovered important differences. Migration into the presumptive caudal branchial arches of the lamprey involves both rostral and caudal movements of neural crest cells that have not been described in gnathostomes, suggesting that barriers that constrain rostrocaudal movement of cranial neural crest cells may have arisen after the agnathan/gnathostome split. Accordingly, neural crest cells from a single axial level contributed to multiple arches and there was extensive mixing between populations. There was no apparent filling of neural crest derivatives in a ventral-to-dorsal order, as has been observed in higher vertebrates, nor did we find evidence of a neural crest contribution to cranial sensory ganglia. These results suggest that migratory constraints and additional neural crest derivatives arose later in gnathostome evolution.

  20. Altered calcium handling and increased contraction force in human embryonic stem cell derived cardiomyocytes following short term dexamethasone exposure

    Energy Technology Data Exchange (ETDEWEB)

    Kosmidis, Georgios; Bellin, Milena; Ribeiro, Marcelo C.; Meer, Berend van; Ward-van Oostwaard, Dorien [Department of Anatomy and Embryology, Leiden University Medical Center, Leiden (Netherlands); Passier, Robert [Department of Anatomy and Embryology, Leiden University Medical Center, Leiden (Netherlands); MIRA, University of Twente (Netherlands); Tertoolen, Leon G.J.; Mummery, Christine L. [Department of Anatomy and Embryology, Leiden University Medical Center, Leiden (Netherlands); Casini, Simona, E-mail: s.casini@amc.uva.nl [Department of Anatomy and Embryology, Leiden University Medical Center, Leiden (Netherlands)

    2015-11-27

    One limitation in using human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) for disease modeling and cardiac safety pharmacology is their immature functional phenotype compared with adult cardiomyocytes. Here, we report that treatment of human embryonic stem cell derived cardiomyocytes (hESC-CMs) with dexamethasone, a synthetic glucocorticoid, activated glucocorticoid signaling which in turn improved their calcium handling properties and contractility. L-type calcium current and action potential properties were not affected by dexamethasone but significantly faster calcium decay, increased forces of contraction and sarcomeric lengths, were observed in hESC-CMs after dexamethasone exposure. Activating the glucocorticoid pathway can thus contribute to mediating hPSC-CMs maturation. - Highlights: • Dexamethasone accelerates Ca{sup 2+} transient decay in hESC-CMs. • Dexamethasone enhances SERCA and NCX function in hESC-CMs. • Dexamethasone increases force of contraction and sarcomere length in hESC-CMs. • Dexamethasone does not alter I{sub Ca,L} and action potential characteristics in hESC-CMs.

  1. Defined MicroRNAs Induce Aspects of Maturation in Mouse and Human Embryonic-Stem-Cell-Derived Cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Desy S. Lee

    2015-09-01

    Full Text Available Pluripotent-cell-derived cardiomyocytes have great potential for use in research and medicine, but limitations in their maturity currently constrain their usefulness. Here, we report a method for improving features of maturation in murine and human embryonic-stem-cell-derived cardiomyocytes (m/hESC-CMs. We found that coculturing m/hESC-CMs with endothelial cells improves their maturity and upregulates several microRNAs. Delivering four of these microRNAs, miR-125b-5p, miR-199a-5p, miR-221, and miR-222 (miR-combo, to m/hESC-CMs resulted in improved sarcomere alignment and calcium handling, a more negative resting membrane potential, and increased expression of cardiomyocyte maturation markers. Although this could not fully phenocopy all adult cardiomyocyte characteristics, these effects persisted for two months following delivery of miR-combo. A luciferase assay demonstrated that all four miRNAs target ErbB4, and siRNA knockdown of ErbB4 partially recapitulated the effects of miR-combo. In summary, a combination of miRNAs induced via endothelial coculture improved ESC-CM maturity, in part through suppression of ErbB4 signaling.

  2. Therapeutic potential of CAR-T cell-derived exosomes: a cell-free modality for targeted cancer therapy.

    Science.gov (United States)

    Tang, Xiang-Jun; Sun, Xu-Yong; Huang, Kuan-Ming; Zhang, Li; Yang, Zhuo-Shun; Zou, Dan-Dan; Wang, Bin; Warnock, Garth L; Dai, Long-Jun; Luo, Jie

    2015-12-29

    Chimeric antigen receptor (CAR)-based T-cell adoptive immunotherapy is a distinctively promising therapy for cancer. The engineering of CARs into T cells provides T cells with tumor-targeting capabilities and intensifies their cytotoxic activity through stimulated cell expansion and enhanced cytokine production. As a novel and potent therapeutic modality, there exists some uncontrollable processes which are the potential sources of adverse events. As an extension of this impactful modality, CAR-T cell-derived exosomes may substitute CAR-T cells to act as ultimate attackers, thereby overcoming some limitations. Exosomes retain most characteristics of parent cells and play an essential role in intercellular communications via transmitting their cargo to recipient cells. The application of CAR-T cell-derived exosomes will make this cell-based therapy more clinically controllable as it also provides a cell-free platform to diversify anticancer mediators, which responds effectively to the complexity and volatility of cancer. It is believed that the appropriate application of both cellular and exosomal platforms will make this effective treatment more practicable.

  3. Evaluation of Stem Cell-Derived Red Blood Cells as a Transfusion Product Using a Novel Animal Model.

    Science.gov (United States)

    Shah, Sandeep N; Gelderman, Monique P; Lewis, Emily M A; Farrel, John; Wood, Francine; Strader, Michael Brad; Alayash, Abdu I; Vostal, Jaroslav G

    2016-01-01

    Reliance on volunteer blood donors can lead to transfusion product shortages, and current liquid storage of red blood cells (RBCs) is associated with biochemical changes over time, known as 'the storage lesion'. Thus, there is a need for alternative sources of transfusable RBCs to supplement conventional blood donations. Extracorporeal production of stem cell-derived RBCs (stemRBCs) is a potential and yet untapped source of fresh, transfusable RBCs. A number of groups have attempted RBC differentiation from CD34+ cells. However, it is still unclear whether these stemRBCs could eventually be effective substitutes for traditional RBCs due to potential differences in oxygen carrying capacity, viability, deformability, and other critical parameters. We have generated ex vivo stemRBCs from primary human cord blood CD34+ cells and compared them to donor-derived RBCs based on a number of in vitro parameters. In vivo, we assessed stemRBC circulation kinetics in an animal model of transfusion and oxygen delivery in a mouse model of exercise performance. Our novel, chronically anemic, SCID mouse model can evaluate the potential of stemRBCs to deliver oxygen to tissues (muscle) under resting and exercise-induced hypoxic conditions. Based on our data, stem cell-derived RBCs have a similar biochemical profile compared to donor-derived RBCs. While certain key differences remain between donor-derived RBCs and stemRBCs, the ability of stemRBCs to deliver oxygen in a living organism provides support for further development as a transfusion product.

  4. Immune cell-derived c3 is required for autoimmune diabetes induced by multiple low doses of streptozotocin.

    Science.gov (United States)

    Lin, Marvin; Yin, Na; Murphy, Barbara; Medof, M Edward; Segerer, Stephan; Heeger, Peter S; Schröppel, Bernd

    2010-09-01

    The complement system contributes to autoimmune injury, but its involvement in promoting the development of autoimmune diabetes is unknown. In this study, our goal was to ascertain the role of complement C3 in autoimmune diabetes. Susceptibility to diabetes development after multiple low-dose streptozotocin treatment in wild-type (WT) and C3-deficient mice was analyzed. Bone marrow chimeras, luminex, and quantitative reverse transcription PCR assays were performed to evaluate the phenotypic and immunologic impact of C3 in the development of this diabetes model. Coincident with the induced elevations in blood glucose levels, we documented alternative pathway complement component gene expression within the islets of the diabetic WT mice. When we repeated the experiments with C3-deficient mice, we observed complete resistance to disease, as assessed by the absence of histologic insulitis and the absence of T-cell reactivity to islet antigens. Studies of WT chimeras bearing C3-deficient bone marrow cells showed that bone marrow cell-derived C3, and not serum C3, is involved in the induction of diabetes in this model. The data reveal a key role for immune cell-derived C3 in the pathogenesis of murine multiple low-dose streptozotocin-induced diabetes and support the concept that immune cell mediated diabetes is in part complement-dependent.

  5. Innate immune cell-derived microparticles facilitate hepatocarcinoma metastasis by transferring integrin α(M)β₂ to tumor cells.

    Science.gov (United States)

    Ma, Jingwei; Cai, Wenqian; Zhang, Yi; Huang, Chunmei; Zhang, Huafeng; Liu, Jing; Tang, Ke; Xu, Pingwei; Katirai, Foad; Zhang, Jianmin; He, Wei; Ye, Duyun; Shen, Guan-Xin; Huang, Bo

    2013-09-15

    Mechanisms by which tumor cells metastasize to distant organs still remain enigmatic. Immune cells have been assumed to be the root of metastasis by their fusing with tumor cells. This fusion theory, although interpreting tumor metastasis analogically and intriguingly, is arguable to date. We show in this study an alternative explanation by immune cell-derived microparticles (MPs). Upon stimulation by PMA or tumor cell-derived supernatants, immune cells released membrane-based MPs, which were taken up by H22 tumor cells, leading to tumor cell migration in vitro and metastasis in vivo. The underlying molecular basis was involved in integrin α(M)β₂ (CD11b/CD18), which could be effectively relayed from stimulated innate immune cells to MPs, then to tumor cells. Blocking either CD11b or CD18 led to significant decreases in MP-mediated tumor cell metastasis. This MP-mediated transfer of immune phenotype to tumor cells might also occur in vivo. These findings suggest that tumor cells may usurp innate immune cell phenotypes via MP pathway for their metastasis, providing new insight into tumor metastatic mechanism.

  6. Altered calcium handling and increased contraction force in human embryonic stem cell derived cardiomyocytes following short term dexamethasone exposure

    International Nuclear Information System (INIS)

    Kosmidis, Georgios; Bellin, Milena; Ribeiro, Marcelo C.; Meer, Berend van; Ward-van Oostwaard, Dorien; Passier, Robert; Tertoolen, Leon G.J.; Mummery, Christine L.; Casini, Simona

    2015-01-01

    One limitation in using human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) for disease modeling and cardiac safety pharmacology is their immature functional phenotype compared with adult cardiomyocytes. Here, we report that treatment of human embryonic stem cell derived cardiomyocytes (hESC-CMs) with dexamethasone, a synthetic glucocorticoid, activated glucocorticoid signaling which in turn improved their calcium handling properties and contractility. L-type calcium current and action potential properties were not affected by dexamethasone but significantly faster calcium decay, increased forces of contraction and sarcomeric lengths, were observed in hESC-CMs after dexamethasone exposure. Activating the glucocorticoid pathway can thus contribute to mediating hPSC-CMs maturation. - Highlights: • Dexamethasone accelerates Ca"2"+ transient decay in hESC-CMs. • Dexamethasone enhances SERCA and NCX function in hESC-CMs. • Dexamethasone increases force of contraction and sarcomere length in hESC-CMs. • Dexamethasone does not alter I_C_a_,_L and action potential characteristics in hESC-CMs.

  7. Impaired APP activity and altered Tau splicing in embryonic stem cell-derived astrocytes obtained from an APPsw transgenic minipig

    Directory of Open Access Journals (Sweden)

    Vanessa J. Hall

    2015-10-01

    Full Text Available Animal models of familial juvenile onset of Alzheimer's disease (AD often fail to produce diverse pathological features of the disease by modification of single gene mutations that are responsible for the disease. They can hence be poor models for testing and development of novel drugs. Here, we analyze in vitro-produced stem cells and their derivatives from a large mammalian model of the disease created by overexpression of a single mutant human gene (APPsw. We produced hemizygous and homozygous radial glial-like cells following culture and differentiation of embryonic stem cells (ESCs isolated from embryos obtained from mated hemizygous minipigs. These cells were confirmed to co-express varying neural markers, including NES, GFAP and BLBP, typical of type one radial glial cells (RGs from the subgranular zone. These cells had altered expression of CCND1 and NOTCH1 and decreased expression of several ribosomal RNA genes. We found that these cells were able to differentiate into astrocytes upon directed differentiation. The astrocytes produced had decreased α- and β-secretase activity, increased γ-secretase activity and altered splicing of tau. This indicates novel aspects of early onset mechanisms related to cell renewal and function in familial AD astrocytes. These outcomes also highlight that radial glia could be a potentially useful population of cells for drug discovery, and that altered APP expression and altered tau phosphorylation can be detected in an in vitro model of the disease. Finally, it might be possible to use large mammal models to model familial AD by insertion of only a single mutation.

  8. Effect of leukemia inhibitory factor on long-term propagation of precursor cells derived from rat forebrain subventricular zone and ventral mesencephalon

    DEFF Research Database (Denmark)

    Andersen, Rikke K; Zimmer, Jens; Wahlberg, Lars U

    2008-01-01

    Tissue blocks containing neural precursor cells were isolated from the rat forebrain subventricular zone (SVZ) and ventral mesencephalon (VM) and propagated as neural tissue-spheres (NTS). In the presence of fibroblast growth factor-2 (FGF2) and epidermal growth factor (EGF), SVZ-derived NTS were...... propagated and maintained for more than 6 months with a cell population doubling time of 21.5 days. The replacement of EGF by leukemia inhibitory factor (LIF) resulted in a cell population doubling time of 19.8 days, corresponding to a 10-fold increase in estimated cell numbers over a period of 70 days......, at which point these NTS ceased to grow. In the presence of FGF2 and LIF, VM-derived NTS displayed a cell population doubling time of 24.6 days, which was maintained over a period of more than 200 days. However, when LIF was replaced by EGF, the cell numbers only increased 1.2 fold over 50 days. Using...

  9. Advances of mesenchymal stem cells derived from bone marrow and dental tissue in craniofacial tissue engineering.

    Science.gov (United States)

    Yang, Maobin; Zhang, Hongming; Gangolli, Riddhi

    2014-05-01

    Bone and dental tissues in craniofacial region work as an important aesthetic and functional unit. Reconstruction of craniofacial tissue defects is highly expected to ensure patients to maintain good quality of life. Tissue engineering and regenerative medicine have been developed in the last two decades, and been advanced with the stem cell technology. Bone marrow derived mesenchymal stem cells are one of the most extensively studied post-natal stem cell population, and are widely utilized in cell-based therapy. Dental tissue derived mesenchymal stem cells are a relatively new stem cell population that isolated from various dental tissues. These cells can undergo multilineage differentiation including osteogenic and odontogenic differentiation, thus provide an alternative source of mesenchymal stem cells for tissue engineering. In this review, we discuss the important issues in mesenchymal stem cell biology including the origin and functions of mesenchymal stem cells, compare the properties of these two types of mesenchymal cells, update recent basic research and clinic applications in this field, and address important future challenges.

  10. Leukemia inhibitory factor (LIF) enhances MAP2 + and HUC/D + neurons and influences neurite extension during differentiation of neural progenitors derived from human embryonic stem cells.

    Science.gov (United States)

    Leukemia Inhibitory Factor (L1F), a member of the Interleukin 6 cytokine family, has a role in differentiation of Human Neural Progenitor (hNP) cells in vitro. hNP cells, derived from Human Embryonic Stem (hES) cells, have an unlimited capacity for self-renewal in monolayer cultu...

  11. Interclonal variations in the molecular karyotype of Trypanosoma cruzi: chromosome rearrangements in a single cell-derived clone of the G strain.

    Science.gov (United States)

    Lima, Fabio Mitsuo; Souza, Renata Torres; Santori, Fábio Rinaldo; Santos, Michele Fernandes; Cortez, Danielle Rodrigues; Barros, Roberto Moraes; Cano, Maria Isabel; Valadares, Helder Magno Silva; Macedo, Andréa Mara; Mortara, Renato Arruda; da Silveira, José Franco

    2013-01-01

    Trypanosoma cruzi comprises a pool of populations which are genetically diverse in terms of DNA content, growth and infectivity. Inter- and intra-strain karyotype heterogeneities have been reported, suggesting that chromosomal rearrangements occurred during the evolution of this parasite. Clone D11 is a single-cell-derived clone of the T. cruzi G strain selected by the minimal dilution method and by infecting Vero cells with metacyclic trypomastigotes. Here we report that the karyotype of clone D11 differs from that of the G strain in both number and size of chromosomal bands. Large chromosomal rearrangement was observed in the chromosomes carrying the tubulin loci. However, most of the chromosome length polymorphisms were of small amplitude, and the absence of one band in clone D11 in relation to its reference position in the G strain could be correlated to the presence of a novel band migrating above or below this position. Despite the presence of chromosomal polymorphism, large syntenic groups were conserved between the isolates. The appearance of new chromosomal bands in clone D11 could be explained by chromosome fusion followed by a chromosome break or interchromosomal exchange of large DNA segments. Our results also suggest that telomeric regions are involved in this process. The variant represented by clone D11 could have been induced by the stress of the cloning procedure or could, as has been suggested for Leishmania infantum, have emerged from a multiclonal, mosaic parasite population submitted to frequent DNA amplification/deletion events, leading to a 'mosaic' structure with different individuals having differently sized versions of the same chromosomes. If this is the case, the variant represented by clone D11 would be better adapted to survive the stress induced by cloning, which includes intracellular development in the mammalian cell. Karyotype polymorphism could be part of the T. cruzi arsenal for responding to environmental pressure.

  12. Cell-derived microparticles: new targets in the therapeutic management of disease.

    Science.gov (United States)

    Roseblade, Ariane; Luk, Frederick; Rawling, Tristan; Ung, Alison; Grau, Georges E R; Bebawy, Mary

    2013-01-01

    Intercellular communication is essential to maintain vital physiological activities and to regulate the organism's phenotype. There are a number of ways in which cells communicate with one another. This can occur via autocrine signaling, endocrine signaling or by the transfer of molecular mediators across gap junctions. More recently communication via microvesicular shedding has gained important recognition as a significant pathway by which cells can coordinate the spread and dominance of selective traits within a population. Through this communication apparatus, cells can now acquire and secure a survival advantage, particularly in the context of malignant disease. This review aims to highlight some of the functions and implications of microparticles in physiology of various disease states, and present a novel therapeutic strategy through the regulation of microparticle production.

  13. Comparison of intraspinal and intrathecal implantation of induced pluripotent stem cell-derived neural precursors for the treatment of spinal cord injury in rats

    Czech Academy of Sciences Publication Activity Database

    Amemori, Takashi; Růžička, Jiří; Romanyuk, Nataliya; Jhanwar-Uniyal, M.; Syková, Eva; Jendelová, Pavla

    2015-01-01

    Roč. 6, Dec (2015), s. 257 ISSN 1757-6512 R&D Projects: GA MŠk(CZ) LH12024 Institutional support: RVO:68378041 Keywords : spinal cord injury * human induced pluripotent stem cells * cell therapy * cell application route Subject RIV: FH - Neurology Impact factor: 4.504, year: 2015

  14. Automated grouping of action potentials of human embryonic stem cell-derived cardiomyocytes.

    Science.gov (United States)

    Gorospe, Giann; Zhu, Renjun; Millrod, Michal A; Zambidis, Elias T; Tung, Leslie; Vidal, Rene

    2014-09-01

    Methods for obtaining cardiomyocytes from human embryonic stem cells (hESCs) are improving at a significant rate. However, the characterization of these cardiomyocytes (CMs) is evolving at a relatively slower rate. In particular, there is still uncertainty in classifying the phenotype (ventricular-like, atrial-like, nodal-like, etc.) of an hESC-derived cardiomyocyte (hESC-CM). While previous studies identified the phenotype of a CM based on electrophysiological features of its action potential, the criteria for classification were typically subjective and differed across studies. In this paper, we use techniques from signal processing and machine learning to develop an automated approach to discriminate the electrophysiological differences between hESC-CMs. Specifically, we propose a spectral grouping-based algorithm to separate a population of CMs into distinct groups based on the similarity of their action potential shapes. We applied this method to a dataset of optical maps of cardiac cell clusters dissected from human embryoid bodies. While some of the nine cell clusters in the dataset are presented with just one phenotype, the majority of the cell clusters are presented with multiple phenotypes. The proposed algorithm is generally applicable to other action potential datasets and could prove useful in investigating the purification of specific types of CMs from an electrophysiological perspective.

  15. The homeobox gene Hex regulates hepatocyte differentiation from embryonic stem cell-derived endoderm.

    Science.gov (United States)

    Kubo, Atsushi; Kim, Yon Hui; Irion, Stefan; Kasuda, Shogo; Takeuchi, Mitsuaki; Ohashi, Kazuo; Iwano, Masayuki; Dohi, Yoshiko; Saito, Yoshihiko; Snodgrass, Ralph; Keller, Gordon

    2010-02-01

    We investigated the role of the hematopoietically expressed homeobox (Hex) in the differentiation and development of hepatocytes within embryonic stem cell (ESC)-derived embryoid bodies (EBs). Analyses of hepatic endoderm derived from Hex(-/-) EBs revealed a dramatic reduction in the levels of albumin (Alb) and alpha-fetoprotein (Afp) expression. In contrast, stage-specific forced expression of Hex in EBs from wild-type ESCs led to the up-regulation of Alb and Afp expression and secretion of Alb and transferrin. These inductive effects were restricted to c-kit(+) endoderm-enriched EB-derived populations, suggesting that Hex functions at the level of hepatic specification of endoderm in this model. Microarray analysis revealed that Hex regulated the expression of a broad spectrum of hepatocyte-related genes, including fibrinogens, apolipoproteins, and cytochromes. When added to the endoderm-induced EBs, bone morphogenetic protein 4 acted synergistically with Hex in the induction of expression of Alb, Afp, carbamoyl phosphate synthetase, transcription factor 1, and CCAAT/enhancer binding protein alpha. These findings indicate that Hex plays a pivotal role during induction of liver development from endoderm in this in vitro model and suggest that this strategy may provide important insight into the generation of functional hepatocytes from ESCs.

  16. Current View on Osteogenic Differentiation Potential of Mesenchymal Stromal Cells Derived from Placental Tissues.

    Science.gov (United States)

    Kmiecik, Gabriela; Spoldi, Valentina; Silini, Antonietta; Parolini, Ornella

    2015-08-01

    Mesenchymal stromal cells (MSC) isolated from human term placental tissues possess unique characteristics, including their peculiar immunomodulatory properties and their multilineage differentiation potential. The osteogenic differentiation capacity of placental MSC has been widely disputed, and continues to be an issue of debate. This review will briefly discuss the different MSC populations which can be obtained from different regions of human term placenta, along with their unique properties, focusing specifically on their osteogenic differentiation potential. We will present the strategies used to enhance osteogenic differentiation potential in vitro, such as through the selection of subpopulations more prone to differentiate, the modification of the components of osteo-inductive medium, and even mechanical stimulation. Accordingly, the applications of three-dimensional environments in vitro and in vivo, such as non-synthetic, polymer-based, and ceramic scaffolds, will also be discussed, along with results obtained from pre-clinical studies of placental MSC for the regeneration of bone defects and treatment of bone-related diseases.

  17. Concise review: stem cell-derived erythrocytes as upcoming players in blood transfusion.

    Science.gov (United States)

    Zeuner, Ann; Martelli, Fabrizio; Vaglio, Stefania; Federici, Giulia; Whitsett, Carolyn; Migliaccio, Anna Rita

    2012-08-01

    Blood transfusions have become indispensable to treat the anemia associated with a variety of medical conditions ranging from genetic disorders and cancer to extensive surgical procedures. In developed countries, the blood supply is generally adequate. However, the projected decline in blood donor availability due to population ageing and the difficulty in finding rare blood types for alloimmunized patients indicate a need for alternative red blood cell (RBC) transfusion products. Increasing knowledge of processes that govern erythropoiesis has been translated into efficient procedures to produce RBC ex vivo using primary hematopoietic stem cells, embryonic stem cells, or induced pluripotent stem cells. Although in vitro-generated RBCs have recently entered clinical evaluation, several issues related to ex vivo RBC production are still under intense scrutiny: among those are the identification of stem cell sources more suitable for ex vivo RBC generation, the translation of RBC culture methods into clinical grade production processes, and the development of protocols to achieve maximal RBC quality, quantity, and maturation. Data on size, hemoglobin, and blood group antigen expression and phosphoproteomic profiling obtained on erythroid cells expanded ex vivo from a limited number of donors are presented as examples of the type of measurements that should be performed as part of the quality control to assess the suitability of these cells for transfusion. New technologies for ex vivo erythroid cell generation will hopefully provide alternative transfusion products to meet present and future clinical requirements. Copyright © 2012 AlphaMed Press.

  18. Sex Differences in Maturation of Human Embryonic Stem Cell-Derived β Cells in Mice.

    Science.gov (United States)

    Saber, Nelly; Bruin, Jennifer E; O'Dwyer, Shannon; Schuster, Hellen; Rezania, Alireza; Kieffer, Timothy J

    2018-04-01

    Pancreatic progenitors derived from human embryonic stem cells (hESCs) are now in clinical trials for insulin replacement in patients with type 1 diabetes. Animal studies indicate that pancreatic progenitor cells can mature into a mixed population of endocrine cells, including glucose-responsive β cells several months after implantion. However, it remains unclear how conditions in the recipient may influence the maturation and ultimately the function of these hESC-derived cells. Here, we investigated the effects of (1) pregnancy on the maturation of human stage 4 (S4) pancreatic progenitor cells and (2) the impact of host sex on both S4 cells and more mature stage 7 (S7) pancreatic endocrine cells implanted under the kidney capsule of immunodeficient SCID-beige mice. Pregnancy led to increased proliferation of endogenous pancreatic β cells, but did not appear to affect proliferation or maturation of S4 cells at midgestation. Interestingly, S4 and S7 cells both acquired glucose-stimulated C-peptide secretion in females before males. Moreover, S4 cells lowered fasting blood glucose levels in females sooner than in males, whereas the responses with S7 cells were similar. These data indicate that the host sex may impact the maturation of hESC-derived cells in vivo and that this effect can be minimized by more advanced differentiation of the cells before implantation.

  19. Apoptotic Tumor Cell-Derived Extracellular Vesicles as Important Regulators of the Onco-Regenerative Niche

    Directory of Open Access Journals (Sweden)

    Christopher D. Gregory

    2018-05-01

    Full Text Available Cells undergoing apoptosis produce heterogeneous populations of membrane delimited extracellular vesicles (Apo-EVs which vary not only in size—from tens of nanometers to several microns—but also in molecular composition and cargo. Apo-EVs carry a variety of potentially biologically active components, including small molecules, proteins, and nucleic acids. Larger forms of Apo-EVs, commonly termed “apoptotic bodies,” can carry organelles, such as mitochondria and nuclear fragments. Molecules displayed on the surface of extracellular vesicles (EVs can contribute substantially to their size, as well as their functions. Thus far, relatively little is known of the functional significance of Apo-EVs apart from their roles in fragmentation of dying cells and indicated immunomodulatory activities. Here, we discuss EV production by dying tumor cells and consider the possible roles of Apo-EVs in a cell death-driven sector of the tumor microenvironment known as the onco-regenerative niche (ORN. We propose that tumor-derived Apo-EVs are significant vehicles of the ORN, functioning as critical intercellular communicators that activate oncogenic tissue repair and regeneration pathways. We highlight important outstanding questions and suggest that Apo-EVs may harbor novel therapeutic targets.

  20. Controversies in Cardiovascular Research: Induced pluripotent stem cell-derived cardiomyocytes – boutique science or valuable arrhythmia model?

    Science.gov (United States)

    Knollmann, Björn C

    2013-01-01

    As part of the series on Controversies in Cardiovascular Research, the article reviews the strengths and limitations of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) as models of cardiac arrhythmias. Specifically, the article attempts to answer the following questions: Which clinical arrhythmias can be modeled by iPSC-CM? How well can iPSC-CM model adult ventricular myocytes? What are the strengths and limitations of published iPSC-CM arrhythmia models? What new mechanistic insight has been gained? What is the evidence that would support using iPSC-CM to personalize anti-arrhythmic drug therapy? The review also discusses the pros and cons of using the iPSC-CM technology for modeling specific genetic arrhythmia disorders such as long QT syndrome, Brugada Syndrome or Catecholaminergic Polymorphic Ventricular Tachycardia. PMID:23569106

  1. An Abbreviated Protocol for In Vitro Generation of Functional Human Embryonic Stem Cell-Derived Beta-Like Cells

    DEFF Research Database (Denmark)

    Massumi, Mohammad; Pourasgari, Farzaneh; Nalla, Amarnadh

    2016-01-01

    developed an abbreviated five-stage protocol (25-30 days) to generate human Embryonic Stem Cell-Derived Beta-like Cells (ES-DBCs). We showed that Geltrex, as an extracellular matrix, could support the generation of ES-DBCs more efficiently than that of the previously described culture systems......The ability to yield glucose-responsive pancreatic beta-cells from human pluripotent stem cells in vitro will facilitate the development of the cell replacement therapies for the treatment of Type 1 Diabetes. Here, through the sequential in vitro targeting of selected signaling pathways, we have...... positive cells, 1% insulin and glucagon positive cells and 30% insulin and NKX6.1 co-expressing cells. Functionally, ES-DBCs were responsive to high glucose in static incubation and perifusion studies, and could secrete insulin in response to successive glucose stimulations. Mitochondrial metabolic flux...

  2. Matrigel Mattress: A Method for the Generation of Single Contracting Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

    Science.gov (United States)

    Feaster, Tromondae K; Cadar, Adrian G; Wang, Lili; Williams, Charles H; Chun, Young Wook; Hempel, Jonathan E; Bloodworth, Nathaniel; Merryman, W David; Lim, Chee Chew; Wu, Joseph C; Knollmann, Björn C; Hong, Charles C

    2015-12-04

    The lack of measurable single-cell contractility of human-induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) currently limits the utility of hiPSC-CMs for evaluating contractile performance for both basic research and drug discovery. To develop a culture method that rapidly generates contracting single hiPSC-CMs and allows quantification of cell shortening with standard equipment used for studying adult CMs. Single hiPSC-CMs were cultured for 5 to 7 days on a 0.4- to 0.8-mm thick mattress of undiluted Matrigel (mattress hiPSC-CMs) and compared with hiPSC-CMs maintained on a control substrate (method enables the rapid generation of robustly contracting hiPSC-CMs and enhances maturation. This new method allows quantification of contractile performance at the single-cell level, which should be valuable to disease modeling, drug discovery, and preclinical cardiotoxicity testing. © 2015 American Heart Association, Inc.

  3. Transformation of cell-derived microparticles into quantum-dot-labeled nanovectors for antitumor siRNA delivery.

    Science.gov (United States)

    Chen, Gang; Zhu, Jun-Yi; Zhang, Zhi-Ling; Zhang, Wei; Ren, Jian-Gang; Wu, Min; Hong, Zheng-Yuan; Lv, Cheng; Pang, Dai-Wen; Zhao, Yi-Fang

    2015-01-12

    Cell-derived microparticles (MPs) have been recently recognized as critical intercellular information conveyors. However, further understanding of their biological behavior and potential application has been hampered by the limitations of current labeling techniques. Herein, a universal donor-cell-assisted membrane biotinylation strategy was proposed for labeling MPs by skillfully utilizing the natural membrane phospholipid exchange of their donor cells. This innovative strategy conveniently led to specific, efficient, reproducible, and biocompatible quantum dot (QD) labeling of MPs, thereby reliably conferring valuable traceability on MPs. By further loading with small interference RNA, QD-labeled MPs that had inherent cell-targeting and biomolecule-conveying ability were successfully employed for combined bioimaging and tumor-targeted therapy. This study provides the first reliable and biofriendly strategy for transforming biogenic MPs into functionalized nanovectors. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Integrated Analysis of Contractile Kinetics, Force Generation, and Electrical Activity in Single Human Stem Cell-Derived Cardiomyocytes

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    Jan David Kijlstra

    2015-12-01

    Full Text Available The quantitative analysis of cardiomyocyte function is essential for stem cell-based approaches for the in vitro study of human cardiac physiology and pathophysiology. We present a method to comprehensively assess the function of single human pluripotent stem cell-derived cardiomyocyte (hPSC-CMs through simultaneous quantitative analysis of contraction kinetics, force generation, and electrical activity. We demonstrate that statistical analysis of movies of contracting hPSC-CMs can be used to quantify changes in cellular morphology over time and compute contractile kinetics. Using a biomechanical model that incorporates substrate stiffness, we calculate cardiomyocyte force generation at single-cell resolution and validate this approach with conventional traction force microscopy. The addition of fluorescent calcium indicators or membrane potential dyes allows the simultaneous analysis of contractility and calcium handling or action potential morphology. Accordingly, our approach has the potential for broad application in the study of cardiac disease, drug discovery, and cardiotoxicity screening.

  5. Andrographolide - A promising therapeutic agent, negatively regulates glial cell derived neurodegeneration of prefrontal cortex, hippocampus and working memory impairment.

    Science.gov (United States)

    Das, Sudeshna; Mishra, K P; Ganju, Lilly; Singh, S B

    2017-12-15

    Over activation of glial cell derived innate immune factors induces neuro-inflammation that results in neurodegenerative disease, like working memory impairment. In this study, we have investigated the role of andrographolide, a major constituent of Andrographis paniculata plant, in reduction of reactive glial cell derived working memory impairment. Real time PCR, Western bloting, flow cytometric and immunofluorescence studies demonstrated that andrographolide inhibited lipopolysaccharide (LPS)-induced overexpression of HMGB1, TLR4, NFκB, COX-2, iNOS, and release of inflammatory mediators in primary mix glial culture, adult mice prefrontal cortex and hippocampus region. Active microglial and reactive astrocytic makers were also downregulated after andrographolide treatment. Andrographolide suppressed overexpression of microglial MIP-1α, P2X7 receptor and its downstream signaling mediators including-inflammasome NLRP3, caspase1 and mature IL-1β. Furthermore, in vivo maze studies suggested that andrographolide treatment reversed LPS-induced behavioural and working memory disturbances including regulation of expression of protein markers like PKC, p-CREB, amyloid beta, APP, p-tau, synapsin and PSD-95. Andrographolide, by lowering expression of pro apoptotic genes and enhancing the expression of anti-apoptotic gene showed its anti-apoptotic nature that in turn reduces neurodegeneration. Morphology studies using Nissl and FJB staining also showed the neuroprotective effect of andrographolide in the prefrontal cortex region. The above studies indicated that andrographolide prevented neuroinflammation-associated neurodegeneration and improved synaptic plasticity markers in cortical as well as hippocampal region which suggests that andrographolide could be a novel pharmacological countermeasure for the treatment of neuroinflammation and neurological disorders related to memory impairment. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Unique proliferation response in odontoblastic cells derived from human skeletal muscle stem cells by cytokine-induced matrix metalloproteinase-3

    International Nuclear Information System (INIS)

    Ozeki, Nobuaki; Hase, Naoko; Kawai, Rie; Yamaguchi, Hideyuki; Hiyama, Taiki; Kondo, Ayami; Nakata, Kazuhiko; Mogi, Makio

    2015-01-01

    A pro-inflammatory cytokine mixture (CM: interleukin (IL)-1β, tumor necrosis factor-α and interferon-γ) and IL-1β-induced matrix metalloproteinase (MMP)-3 activity have been shown to increase the proliferation of rat dental pulp cells and murine stem cell-derived odontoblast-like cells. This suggests that MMP-3 may regulate wound healing and regeneration in the odontoblast-rich dental pulp. Here, we determined whether these results can be extrapolated to human dental pulp by investigating the effects of CM-induced MMP-3 up-regulation on the proliferation and apoptosis of purified odontoblast-like cells derived from human skeletal muscle stem cells. We used siRNA to specifically reduce MMP-3 expression. We found that CM treatment increased MMP-3 mRNA and protein levels as well as MMP-3 activity. Cell proliferation was also markedly increased, with no changes in apoptosis, upon treatment with CM and following the application of exogenous MMP-3. Endogenous tissue inhibitors of metalloproteinases were constitutively expressed during all experiments and unaffected by MMP-3. Although treatment with MMP-3 siRNA suppressed cell proliferation, it also unexpectedly increased apoptosis. This siRNA-mediated increase in apoptosis could be reversed by exogenous MMP-3. These results demonstrate that cytokine-induced MMP-3 activity regulates cell proliferation and suppresses apoptosis in human odontoblast-like cells. - Highlights: • Pro-inflammatory cytokines induce MMP-3 activity in human odontoblast-like cells. • Increased MMP-3 activity can promote cell proliferation in odontoblasts. • Specific loss of MMP-3 increases apoptosis in odontoblasts. • MMP-3 has potential as a promising new target for pupal repair and regeneration

  7. The in Vitro Assessment of Biochemical Factors in Hepatocyte like Cells Derived from Umbilical Cord Blood Stem Cells

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    A KHoramroodi

    2009-10-01

    Full Text Available Introduction & Objective: Umbilical cord blood (UCB is a source of Hematopoietic Stem Cells (HSC and progenitor cells that can reconstitute the hematopoietic system in patients with malignant and nonmalignant disorders. Mesenchymal stem cell-derived from umbilical cord blood (UCB have been differentiated to some kind of cells, such as osteobblast, adipoblast and chondroblast in Vitro. This study examined the differentiation of Umbilical Cord Blood (UCB derived stem cells to functional hepatocytes. Materials & Methods: The present study was an experimental study which was carried out in the Payam-e-Noor University of Tehran in cooperation with Hamedan University of Medical Sciences in 2008. Umbilical cord blood (UCB was obtained from Fatemieh hospital (Hamadan, Iran. Stem cells were isolated from the cord blood by combining density gradient centrifugation with plastic adherence. When the isolated cells reached 80% confluence, they differentiated to hepatocyte like cells. The medium which was used was consists of DMEM and 10% Fetal Bovine Serum (FBS supplemented with 20 ng/mL Hepatocyte Growth Factor (HGF, 10 ng/mL basic Fibroblast Growth Factor (bFGF and 20 ng/mL Oncostatin M (OSM.The medium was changed every 3 days and stored for Albumin (ALB, Alpha Fetoprotein (AFP, Alkaline Phosphatase (ALP, and urea assay. Finally PAS stain was done to study Glycogen storage in the differentiated cell. Results: Measurement of biochemical factors in different days showed that concentration of albumin (ALB, alpha fetoprotein (AFP, alkaline phosphatase (ALP, and Urea gradually increased. Also, PAS staining showed the storage of glycogen in these cells. Conclusion: Stem cell-derived from human umbilical cord blood (HUCB is a new source of cell types for cell transplantation therapy of hepatic diseases and under certain conditions these cells can differentiate into liver cells.

  8. Polyphosphate induces matrix metalloproteinase-3-mediated proliferation of odontoblast-like cells derived from induced pluripotent stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Ozeki, Nobuaki; Hase, Naoko; Yamaguchi, Hideyuki; Hiyama, Taiki; Kawai, Rie [Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya, Aichi 464-8651 (Japan); Kondo, Ayami [Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto, Chikusa-ku, Nagoya 464-8650 (Japan); Nakata, Kazuhiko [Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya, Aichi 464-8651 (Japan); Mogi, Makio, E-mail: makio@dpc.agu.ac.jp [Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto, Chikusa-ku, Nagoya 464-8650 (Japan)

    2015-05-01

    Inorganic polyphosphate [Poly(P)] may represent a physiological source of phosphate and has the ability to induce bone differentiation in osteoblasts. We previously reported that cytokine-induced matrix metalloproteinase (MMP)-3 accelerates the proliferation of purified odontoblast-like cells. In this study, MMP-3 small interfering RNA (siRNA) was transfected into odontoblast-like cells derived from induced pluripotent stem cells to investigate whether MMP-3 activity is induced by Poly(P) and/or is associated with cell proliferation and differentiation into odontoblast-like cells. Treatment with Poly(P) led to an increase in both cell proliferation and additional odontoblastic differentiation. Poly(P)-treated cells showed a small but significant increase in dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1) mRNA expression, which are markers of mature odontoblasts. The cells also acquired additional odontoblast-specific properties including adoption of an odontoblastic phenotype typified by high alkaline phosphatase (ALP) activity and a calcification capacity. In addition, Poly(P) induced expression of MMP-3 mRNA and protein, and increased MMP-3 activity. MMP-3 siRNA-mediated disruption of the expression of these effectors potently suppressed the expression of odontoblastic biomarkers ALP, DSPP, and DMP-1, and blocked calcification. Interestingly, upon siRNA-mediated silencing of MMP-3, we noted a potent and significant decrease in cell proliferation. Using specific siRNAs, we revealed that a unique signaling cascade, Poly(P)→MMP-3→DSPP and/or DMP-1, was intimately involved in the proliferation of odontoblast-like cells. - Highlights: • Polyphosphate increases proliferation of iPS cell-derived odontoblast-like cells. • Polyphosphate-induced MMP-3 results in an increase of cell proliferation. • Induced cell proliferation involves MMP-3, DSPP, and/or DMP-1 sequentially. • Induced MMP-3 also results in an increase of odontoblastic

  9. Unique proliferation response in odontoblastic cells derived from human skeletal muscle stem cells by cytokine-induced matrix metalloproteinase-3

    Energy Technology Data Exchange (ETDEWEB)

    Ozeki, Nobuaki; Hase, Naoko; Kawai, Rie; Yamaguchi, Hideyuki; Hiyama, Taiki [Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya 464-8651, Aichi (Japan); Kondo, Ayami [Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto, Chikusa-ku, Nagoya 464-8650 (Japan); Nakata, Kazuhiko [Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya 464-8651, Aichi (Japan); Mogi, Makio, E-mail: makio@dpc.agu.ac.jp [Department of Medicinal Biochemistry, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto, Chikusa-ku, Nagoya 464-8650 (Japan)

    2015-02-01

    A pro-inflammatory cytokine mixture (CM: interleukin (IL)-1β, tumor necrosis factor-α and interferon-γ) and IL-1β-induced matrix metalloproteinase (MMP)-3 activity have been shown to increase the proliferation of rat dental pulp cells and murine stem cell-derived odontoblast-like cells. This suggests that MMP-3 may regulate wound healing and regeneration in the odontoblast-rich dental pulp. Here, we determined whether these results can be extrapolated to human dental pulp by investigating the effects of CM-induced MMP-3 up-regulation on the proliferation and apoptosis of purified odontoblast-like cells derived from human skeletal muscle stem cells. We used siRNA to specifically reduce MMP-3 expression. We found that CM treatment increased MMP-3 mRNA and protein levels as well as MMP-3 activity. Cell proliferation was also markedly increased, with no changes in apoptosis, upon treatment with CM and following the application of exogenous MMP-3. Endogenous tissue inhibitors of metalloproteinases were constitutively expressed during all experiments and unaffected by MMP-3. Although treatment with MMP-3 siRNA suppressed cell proliferation, it also unexpectedly increased apoptosis. This siRNA-mediated increase in apoptosis could be reversed by exogenous MMP-3. These results demonstrate that cytokine-induced MMP-3 activity regulates cell proliferation and suppresses apoptosis in human odontoblast-like cells. - Highlights: • Pro-inflammatory cytokines induce MMP-3 activity in human odontoblast-like cells. • Increased MMP-3 activity can promote cell proliferation in odontoblasts. • Specific loss of MMP-3 increases apoptosis in odontoblasts. • MMP-3 has potential as a promising new target for pupal repair and regeneration.

  10. Nonanaplastic follicular cell-derived thyroid carcinoma: mitosis and necrosis in long-term follow-up.

    Science.gov (United States)

    Skansing, Daniel Bräuner; Londero, Stefano Christian; Asschenfeldt, Pia; Larsen, Stine Rosenkilde; Godballe, Christian

    2017-06-01

    Nonanaplastic follicular cell-derived thyroid carcinoma (NAFCTC) includes differentiated- (DTC) and poorly differentiated thyroid carcinoma (PDTC). DTC has an excellent prognosis, while PDTC is situated between DTC and anaplastic carcinomas. Short-term studies suggest that PDTC patients diagnosed only on tumor necrosis and/or mitosis have a prognosis similar to those diagnosed according to the TURIN proposal. The purpose of this study was to evaluate prognosis for NAFCTC based on long-term follow-up illuminating the significance of tumor necrosis and mitosis. A cohort of 225 patients with NAFCTC was followed more than 20 years. Age, sex, distant metastasis, histology, tumor size, extrathyroidal invasion, lymph node metastasis, tumor necrosis and mitosis were examined as possible prognostic factors. Median follow-up time for patients alive was 28 years (range 20-43 years). Age, distant metastasis, extrathyroidal invasion, tumor size, tumor necrosis and mitosis were independent prognostic factors in multivariate analysis for overall survival (OS). In disease specific survival (DSS) age was not significant. Using only necrosis and/or mitosis as criteria for PDTC the 5-, 10- and 20-year OS for DTC was 87, 79 and 69%, respectively. In DSS it was 95, 92 and 90%. For PDTC the 5-, 10- and 20-year OS was 57, 40 and 25%, respectively. In DSS it was 71, 55 and 48%. Tumor necrosis and mitosis are highly significant prognostic indicators in analysis of long time survival of nonanaplastic follicular cell-derived thyroid carcinoma indicating that a simplification of the actually used criteria for poorly differentiated carcinomas may be justified.

  11. Extracellular Matrix-Mediated Maturation of Human Pluripotent Stem Cell-Derived Cardiac Monolayer Structure and Electrophysiological Function.

    Science.gov (United States)

    Herron, Todd J; Rocha, Andre Monteiro Da; Campbell, Katherine F; Ponce-Balbuena, Daniela; Willis, B Cicero; Guerrero-Serna, Guadalupe; Liu, Qinghua; Klos, Matt; Musa, Hassan; Zarzoso, Manuel; Bizy, Alexandra; Furness, Jamie; Anumonwo, Justus; Mironov, Sergey; Jalife, José

    2016-04-01

    Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) monolayers generated to date display an immature embryonic-like functional and structural phenotype that limits their utility for research and cardiac regeneration. In particular, the electrophysiological function of hPSC-CM monolayers and bioengineered constructs used to date are characterized by slow electric impulse propagation velocity and immature action potential profiles. Here, we have identified an optimal extracellular matrix for significant electrophysiological and structural maturation of hPSC-CM monolayers. hPSC-CM plated in the optimal extracellular matrix combination have impulse propagation velocities ≈2× faster than previously reported (43.6±7.0 cm/s; n=9) and have mature cardiomyocyte action potential profiles, including hyperpolarized diastolic potential and rapid action potential upstroke velocity (146.5±17.7 V/s; n=5 monolayers). In addition, the optimal extracellular matrix promoted hypertrophic growth of cardiomyocytes and the expression of key mature sarcolemmal (SCN5A, Kir2.1, and connexin43) and myofilament markers (cardiac troponin I). The maturation process reported here relies on activation of integrin signaling pathways: neutralization of β1 integrin receptors via blocking antibodies and pharmacological blockade of focal adhesion kinase activation prevented structural maturation. Maturation of human stem cell-derived cardiomyocyte monolayers is achieved in a 1-week period by plating cardiomyocytes on PDMS (polydimethylsiloxane) coverslips rather than on conventional 2-dimensional cell culture formats, such as glass coverslips or plastic dishes. Activation of integrin signaling and focal adhesion kinase is essential for significant maturation of human cardiac monolayers. © 2016 American Heart Association, Inc.

  12. Evaluation of Stem Cell-Derived Red Blood Cells as a Transfusion Product Using a Novel Animal Model.

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    Sandeep N Shah

    Full Text Available Reliance on volunteer blood donors can lead to transfusion product shortages, and current liquid storage of red blood cells (RBCs is associated with biochemical changes over time, known as 'the storage lesion'. Thus, there is a need for alternative sources of transfusable RBCs to supplement conventional blood donations. Extracorporeal production of stem cell-derived RBCs (stemRBCs is a potential and yet untapped source of fresh, transfusable RBCs. A number of groups have attempted RBC differentiation from CD34+ cells. However, it is still unclear whether these stemRBCs could eventually be effective substitutes for traditional RBCs due to potential differences in oxygen carrying capacity, viability, deformability, and other critical parameters. We have generated ex vivo stemRBCs from primary human cord blood CD34+ cells and compared them to donor-derived RBCs based on a number of in vitro parameters. In vivo, we assessed stemRBC circulation kinetics in an animal model of transfusion and oxygen delivery in a mouse model of exercise performance. Our novel, chronically anemic, SCID mouse model can evaluate the potential of stemRBCs to deliver oxygen to tissues (muscle under resting and exercise-induced hypoxic conditions. Based on our data, stem cell-derived RBCs have a similar biochemical profile compared to donor-derived RBCs. While certain key differences remain between donor-derived RBCs and stemRBCs, the ability of stemRBCs to deliver oxygen in a living organism provides support for further development as a transfusion product.

  13. The role of tumor cell-derived connective tissue growth factor (CTGF/CCN2) in pancreatic tumor growth.

    Science.gov (United States)

    Bennewith, Kevin L; Huang, Xin; Ham, Christine M; Graves, Edward E; Erler, Janine T; Kambham, Neeraja; Feazell, Jonathan; Yang, George P; Koong, Albert; Giaccia, Amato J

    2009-02-01

    Pancreatic cancer is highly aggressive and refractory to existing therapies. Connective tissue growth factor (CTGF/CCN2) is a fibrosis-related gene that is thought to play a role in pancreatic tumor progression. However, CCN2 can be expressed in a variety of cell types, and the contribution of CCN2 derived from either tumor cells or stromal cells as it affects the growth of pancreatic tumors is unknown. Using genetic inhibition of CCN2, we have discovered that CCN2 derived from tumor cells is a critical regulator of pancreatic tumor growth. Pancreatic tumor cells derived from CCN2 shRNA-expressing clones showed dramatically reduced growth in soft agar and when implanted s.c. We also observed a role for CCN2 in the growth of pancreatic tumors implanted orthotopically, with tumor volume measurements obtained by positron emission tomography imaging. Mechanistically, CCN2 protects cells from hypoxia-mediated apoptosis, providing an in vivo selection for tumor cells that express high levels of CCN2. We found that CCN2 expression and secretion was increased in hypoxic pancreatic tumor cells in vitro, and we observed colocalization of CCN2 and hypoxia in pancreatic tumor xenografts and clinical pancreatic adenocarcinomas. Furthermore, we found increased CCN2 staining in clinical pancreatic tumor tissue relative to stromal cells surrounding the tumor, supporting our assertion that tumor cell-derived CCN2 is important for pancreatic tumor growth. Taken together, these data improve our understanding of the mechanisms responsible for pancreatic tumor growth and progression, and also indicate that CCN2 produced by tumor cells represents a viable therapeutic target for the treatment of pancreatic cancer.

  14. Circulating levels of cell-derived microparticles are reduced by mild hypobaric hypoxia: data from a randomised controlled trial.

    Science.gov (United States)

    Ayers, Lisa; Stoewhas, Anne-Christin; Ferry, Berne; Latshang, Tsogyal D; Lo Cascio, Christian M; Sadler, Ross; Stadelmann, Katrin; Tesler, Noemi; Huber, Reto; Achermann, Peter; Bloch, Konrad E; Kohler, Malcolm

    2014-05-01

    Hypoxia is known to induce the release of microparticles in vitro. However, few publications have addressed the role of hypoxia in vivo on circulating levels of microparticles. This randomised, controlled, crossover trial aimed to determine the effect of mild hypoxia on in vivo levels of circulating microparticles in healthy individuals. Blood was obtained from 51 healthy male volunteers (mean age of 26.9 years) at baseline altitude (490 m) and after 24 and 48 h at moderate altitude (2,590 m). The order of altitude exposure was randomised. Flow cytometry was used to assess platelet-poor plasma for levels of circulating microparticles derived from platelets, endothelial cells, leucocytes, granulocytes, monocytes, red blood cells and procoagulant microparticles. Mean (standard deviation) oxygen saturation was significantly lower on the first and second day after arrival at 2,590 m, 91.0 (2.0) and 92.0 (2.0) %, respectively, compared to 490 m, 96 (1.0) %, p microparticles (annexin V+ -221/μl 95 % CI -370.8/-119.0, lactadherin+ -202/μl 95 % CI -372.2/-93.1), platelet-derived microparticles (-114/μl 95 % CI -189.9/-51.0) and red blood cell-derived microparticles (-81.4 μl 95 % CI -109.9/-57.7) after 48 h at moderate altitude was found. Microparticles derived from endothelial cells, granulocytes, monocytes and leucocytes were not significantly altered by exposure to moderate altitude. In healthy male individuals, mild hypobaric hypoxia, induced by a short-term stay at moderate altitude, is associated with lower levels of procoagulant microparticles, platelet-derived microparticles and red blood cell-derived microparticles, suggesting a reduction in thrombotic potential.

  15. Regenerative Medicine, Disease Modelling, and Drug Discovery in Human Pluripotent Stem Cell-Derived Kidney Tissue

    Directory of Open Access Journals (Sweden)

    Navin Gupta

    2017-08-01

    Full Text Available The multitude of research clarifying critical factors in embryonic organ development has been instrumental in human stem cell research. Mammalian organogenesis serves as the archetype for directed differentiation protocols, subdividing the process into a series of distinct intermediate stages that can be chemically induced and monitored for the expression of stage-specific markers. Significant advances over the past few years include established directed differentiation protocols of human embryonic stem cells and human induced pluripotent stem cells (hiPSC into human kidney organoids in vitro. Human kidney tissue in vitro simulates the in vivo response when subjected to nephrotoxins, providing a novel screening platform during drug discovery to facilitate identification of lead candidates, reduce developmental expenditures, and reduce future rates of drug-induced acute kidney injury. Patient-derived hiPSC, which bear naturally occurring DNA mutations, may allow for modelling of human genetic diseases to enable determination of pathological mechanisms and screening for novel therapeutics. In addition, recent advances in genome editing with clustered regularly interspaced short palindromic repeats (CRISPR/Cas9 enable the generation of specific mutations to study genetic disease, with non-mutated lines serving as an ideal isogenic control. The growing population of patients with end-stage kidney disease is a worldwide healthcare problem, with high morbidity and mortality rates, that warrants the discovery of novel forms of renal replacement therapy. Coupling the outlined advances in hiPSC research with innovative bioengineering techniques, such as decellularised kidney and three-dimensional printed scaffolds, may contribute to the development of bioengineered transplantable human kidney tissue as a means of renal replacement therapy.

  16. Isolation and characterization of vascular endothelial cells derived from fetal tooth buds of miniature swine.

    Science.gov (United States)

    Nasu, Masanori; Nakahara, Taka; Tominaga, Noriko; Tamaki, Yuichi; Ide, Yoshiaki; Tachibana, Toshiaki; Ishikawa, Hiroshi

    2013-03-01

    The aim of the present study was to isolate endothelial cells from tooth buds (unerupted deciduous teeth) of miniature swine. Mandibular molar tooth buds harvested from swine fetuses at fetal days 90-110 were cultured in growth medium supplemented with 15% fetal bovine serum in 100-mm culture dishes until the primary cells outgrown from the tooth buds reached confluence. A morphologically defined set of pavement-shaped primary cells were picked up manually with filter paper containing trypsin/ethylenediamine tetraacetic acid solution and transferred to a separate dish. A characterization of the cellular characteristics and a functional analysis of the cultured cells at passages 3 to 5 were performed using immunofluorescence, a reverse transcriptase polymerase chain reaction assay, a tube formation assay, and transmission electron microscopy. The isolated cells grew in a pavement arrangement and showed the characteristics of contact inhibition upon reaching confluence. The population doubling time was ~48 h at passage 3. As shown by immunocytostaining and western blotting with specific antibodies, the cells produced the endothelial marker proteins such as vascular endothelial cadherin, von Willebrand factor, and vascular endothelial growth factor receptor-2. Observation with time-lapse images showed that small groups of cells aggregated and adhered to each other to form tube-like structures. Moreover, as revealed through transmission electron microscopy, these adherent cells had formed junctional complexes. These endothelial cells from the tooth buds of miniature swine are available as cell lines for studies on tube formation and use in regenerative medical science.

  17. Postnatal stem/progenitor cells derived from the dental pulp of adult chimpanzee

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    Fillos Dimitri

    2008-04-01

    Full Text Available Background Chimpanzee dental pulp stem/stromal cells (ChDPSCs are very similar to human bone marrow derived mesenchymal stem/stromal cells (hBMSCs as demonstrated by the expression pattern of cell surface markers and their multipotent differentiation capability. Results ChDPSCs were isolated from an incisor and a canine of a forty-seven year old female chimpanzee. A homogenous population of ChDPSCs was established in early culture at a high proliferation rate and verified by the expression pattern of thirteen cell surface markers. The ChDPSCs are multipotent and were capable of differentiating into osteogenic, adipogenic and chondrogenic lineages under appropriate in vitro culture conditions. ChDPSCs also express stem cell (Sox-2, Nanog, Rex-1, Oct-4 and osteogenic (Osteonectin, osteocalcin, osteopontin markers, which is comparable to reported results of rhesus monkey BMSCs (rBMSCs, hBMSCs and hDPSCs. Although ChDPSCs vigorously proliferated during the initial phase and gradually decreased in subsequent passages, the telomere length indicated that telomerase activity was not significantly reduced. Conclusion These results demonstrate that ChDPSCs can be efficiently isolated from post-mortem teeth of adult chimpanzees and are multipotent. Due to the almost identical genome composition of humans and chimpanzees, there is an emergent need for defining the new role of chimpanzee modeling in comparative medicine. Teeth are easy to recover at necropsy and easy to preserve prior to the retrieval of dental pulp for stem/stromal cells isolation. Therefore, the establishment of ChDPSCs would preserve and maximize the applications of such a unique and invaluable animal model, and could advance the understanding of cellular functions and differentiation control of adult stem cells in higher primates.

  18. Postnatal stem/progenitor cells derived from the dental pulp of adult chimpanzee.

    Science.gov (United States)

    Cheng, Pei-Hsun; Snyder, Brooke; Fillos, Dimitri; Ibegbu, Chris C; Huang, Anderson Hsien-Cheng; Chan, Anthony W S

    2008-04-22

    Chimpanzee dental pulp stem/stromal cells (ChDPSCs) are very similar to human bone marrow derived mesenchymal stem/stromal cells (hBMSCs) as demonstrated by the expression pattern of cell surface markers and their multipotent differentiation capability. ChDPSCs were isolated from an incisor and a canine of a forty-seven year old female chimpanzee. A homogenous population of ChDPSCs was established in early culture at a high proliferation rate and verified by the expression pattern of thirteen cell surface markers. The ChDPSCs are multipotent and were capable of differentiating into osteogenic, adipogenic and chondrogenic lineages under appropriate in vitro culture conditions. ChDPSCs also express stem cell (Sox-2, Nanog, Rex-1, Oct-4) and osteogenic (Osteonectin, osteocalcin, osteopontin) markers, which is comparable to reported results of rhesus monkey BMSCs (rBMSCs), hBMSCs and hDPSCs. Although ChDPSCs vigorously proliferated during the initial phase and gradually decreased in subsequent passages, the telomere length indicated that telomerase activity was not significantly reduced. These results demonstrate that ChDPSCs can be efficiently isolated from post-mortem teeth of adult chimpanzees and are multipotent. Due to the almost identical genome composition of humans and chimpanzees, there is an emergent need for defining the new role of chimpanzee modeling in comparative medicine. Teeth are easy to recover at necropsy and easy to preserve prior to the retrieval of dental pulp for stem/stromal cells isolation. Therefore, the establishment of ChDPSCs would preserve and maximize the applications of such a unique and invaluable animal model, and could advance the understanding of cellular functions and differentiation control of adult stem cells in higher primates.

  19. Electrophysiological Characteristics of Embryonic Stem Cell-Derived Cardiomyocytes are Cell Line-Dependent

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    Tobias Hannes

    2015-01-01

    Full Text Available Background: Modelling of cardiac development, physiology and pharmacology by differentiation of embryonic stem cells (ESCs requires comparability of cardiac differentiation between different ESC lines. To investigate whether the outcome of cardiac differentiation is consistent between different ESC lines, we compared electrophysiological properties of ESC-derived cardiomyocytes (ESC-CMs of different murine ESC lines. Methods: Two wild-type (D3 and R1 and two transgenic ESC lines (D3/aPIG44 and CGR8/AMPIGX-7 were differentiated under identical culture conditions. The transgenic cell lines expressed enhanced green fluorescent protein (eGFP and puromycin-N-acetyltransferase under control of the cardiac specific α-myosin heavy chain (αMHC promoter. Action potentials (APs were recorded using sharp electrodes and multielectrode arrays in beating clusters of ESC-CMs. Results: Spontaneous AP frequency and AP duration (APD as well as maximal upstroke velocity differed markedly between unpurified CMs of the four ESC lines. APD heterogeneity was negligible in D3/aPIG44, moderate in D3 and R1 and extensive in CGR8/AMPIGX-7. Interspike intervals calculated from long-term recordings showed a high degree of variability within and between recordings in CGR8/AMPIGX-7, but not in D3/aPIG44. Purification of the αMHC+ population by puromycin treatment posed only minor changes to APD in D3/aPIG44, but significantly shortened APD in CGR8/AMPIGX-7. Conclusion: Electrophysiological properties of ESC-CMs are strongly cell line-dependent and can be influenced by purification of cardiomyocytes by antibiotic selection. Thus, conclusions on cardiac development, physiology and pharmacology derived from single stem cell lines have to be interpreted carefully.

  20. Epithelial Cell-Derived Secreted and Transmembrane 1a Signals to Activated Neutrophils during Pneumococcal Pneumonia.

    Science.gov (United States)

    Kamata, Hirofumi; Yamamoto, Kazuko; Wasserman, Gregory A; Zabinski, Mary C; Yuen, Constance K; Lung, Wing Yi; Gower, Adam C; Belkina, Anna C; Ramirez, Maria I; Deng, Jane C; Quinton, Lee J; Jones, Matthew R; Mizgerd, Joseph P

    2016-09-01

    Airway epithelial cell responses are critical to the outcome of lung infection. In this study, we aimed to identify unique contributions of epithelial cells during lung infection. To differentiate genes induced selectively in epithelial cells during pneumonia, we compared genome-wide expression profiles from three sorted cell populations: epithelial cells from uninfected mouse lungs, epithelial cells from mouse lungs with pneumococcal pneumonia, and nonepithelial cells from those same infected lungs. Of 1,166 transcripts that were more abundant in epithelial cells from infected lungs compared with nonepithelial cells from the same lungs or from epithelial cells of uninfected lungs, 32 genes were identified as highly expressed secreted products. Especially strong signals included two related secreted and transmembrane (Sectm) 1 genes, Sectm1a and Sectm1b. Refinement of sorting strategies suggested that both Sectm1 products were induced predominantly in conducting airway epithelial cells. Sectm1 was induced during the early stages of pneumococcal pneumonia, and mutation of NF-κB RelA in epithelial cells did not diminish its expression. Instead, type I IFN signaling was necessary and sufficient for Sectm1 induction in lung epithelial cells, mediated by signal transducer and activator of transcription 1. For target cells, Sectm1a bound to myeloid cells preferentially, in particular Ly6G(bright)CD11b(bright) neutrophils in the infected lung. In contrast, Sectm1a did not bind to neutrophils from uninfected lungs. Sectm1a increased expression of the neutrophil-attracting chemokine CXCL2 by neutrophils from the infected lung. We propose that Sectm1a is an epithelial product that sustains a positive feedback loop amplifying neutrophilic inflammation during pneumococcal pneumonia.

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

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    Boku, Shuken, E-mail: shuboku@med.hokudai.ac.jp [Department of Psychiatry, Hokkaido University Graduate School of Medicine, Sapporo (Japan); Nakagawa, Shin [Department of Psychiatry, Hokkaido University Graduate School of Medicine, Sapporo (Japan); Takamura, Naoki [Pharmaceutical Laboratories, Dainippon Sumitomo Pharma Co. Ltd., Osaka (Japan); Kato, Akiko [Department of Psychiatry, Hokkaido University Graduate School of Medicine, Sapporo (Japan); Takebayashi, Minoru [Department of Psychiatry, National Hospital Organization Kure Medical Center, Kure (Japan); Hisaoka-Nakashima, Kazue [Department of Pharmacology, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima (Japan); Omiya, Yuki; Inoue, Takeshi; Kusumi, Ichiro [Department of Psychiatry, Hokkaido University Graduate School of Medicine, Sapporo (Japan)

    2013-05-17

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

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

    International Nuclear Information System (INIS)

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

    2013-01-01

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

  3. Vanadate impedes adipogenesis in mesenchymal stem cells derived from different depots within bone.

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    Frans Alexander Jacobs

    2016-08-01

    Full Text Available Glucocorticoid induced osteoporosis (GIO is associated with an increase in bone marrow adiposity which skews the differentiation of mesenchymal stem cell (MSC progenitors away from osteoblastogenesis and towards adipogenesis. We have previously found that vanadate, a non-specific protein tyrosine phosphatase inhibitor, prevents GIO in rats, but it was unclear whether vanadate directly influenced adipogenesis in bone-derived MSCs. For the present study, we investigated the effect of vanadate on adipogenesis in primary rat MSCs derived from bone marrow (bmMSCs and from the proximal end of the femur (pfMSCs. By passage 3 after isolation, both cell populations expressed the MSC cell surface markers CD90 and CD106, but not the haematopoietic marker CD45. However, although variable, expression of the fibroblast marker CD26 was higher in pfMSCs than in bmMSCs. Differentiation studies using osteogenic and adipogenic induction media (OM and AM, respectively demonstrated that pfMSCs rapidly accumulated lipid droplets within 1 week of exposure to AM, while bmMSCs isolated from the same femur only formed lipid droplets after 3 weeks of AM treatment. Conversely, pfMSCs exposed to OM produced mineralized extracellular matrix (ECM after 3 weeks, compared to 1 week for OM-treated bmMSCs. Vanadate (10 µM added to AM resulted in a significant reduction in AM-induced intracellular lipid accumulation and expression of adipogenic gene markers (PPARγ2, aP2, adipsin in both pfMSCs and bmMSCs. Pharmacological concentrations of glucocorticoids (1 µM alone did not induce lipid accumulation in either bmMSCs or pfMSCs, but resulted in significant cell death in pfMSCs. Our findings demonstrate the existence of at least two fundamentally different MSC depots within the femur, and highlights the presence of MSCs capable of rapid adipogenesis within the proximal femur, an area prone to osteoporotic fractures. In addition, our results suggest that the increased bone marrow

  4. Isolation and cellular properties of mesenchymal cells derived from the decidua of human term placenta.

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    Kanematsu, Daisuke; Shofuda, Tomoko; Yamamoto, Atsuyo; Ban, Chiaki; Ueda, Takafumi; Yamasaki, Mami; Kanemura, Yonehiro

    2011-09-01

    The clinical promise of cell-based therapies is generally recognized, and has driven an intense search for good cell sources. In this study, we isolated plastic-adherent cells from human term decidua vera, called decidua-derived-mesenchymal cells (DMCs), and compared their properties with those of bone marrow-derived-mesenchymal stem cells (BM-MSCs). The DMCs strongly expressed the mesenchymal cell marker vimentin, but not cytokeratin 19 or HLA-G, and had a high proliferative potential. That is, they exhibited a typical fibroblast-like morphology for over 30 population doublings. Cells phenotypically identical to the DMCs were identified in the decidua vera, and genotyping confirmed that the DMCs were derived from the maternal components of the fetal adnexa. Flow cytometry analysis showed that the expression pattern of CD antigens on the DMCs was almost identical to that on BM-MSCs, but some DMCs expressed the CD45 antigen, and over 50% of them also expressed anti-fibroblast antigen. In vitro, the DMCs showed good differentiation into chondrocytes and moderate differentiation into adipocytes, but scant evidence of osteogenesis, compared with the BM-MSCs. Gene expression analysis showed that, compared with BM-MSCs, the DMCs expressed higher levels of TWIST2 and RUNX2 (which are associated with early mesenchymal development and/or proliferative capacity), several matrix metalloproteinases (MMP1, 3, 10, and 12), and cytokines (BMP2 and TGFB2), and lower levels of MSX2, interleukin 26, and HGF. Although DMCs did not show the full multipotency of BM-MSCs, their higher proliferative ability indicates that their cultivation would require less maintenance. Furthermore, the use of DMCs avoids the ethical concerns associated with the use of embryonic tissues, because they are derived from the maternal portion of the placenta, which is otherwise discarded. Thus, the unique properties of DMCs give them several advantages for clinical use, making them an interesting and

  5. Quantitative Analysis of Human Pluripotency and Neural Specification by In-Depth (PhosphoProteomic Profiling

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    Ilyas Singec

    2016-09-01

    Full Text Available Controlled differentiation of human embryonic stem cells (hESCs can be utilized for precise analysis of cell type identities during early development. We established a highly efficient neural induction strategy and an improved analytical platform, and determined proteomic and phosphoproteomic profiles of hESCs and their specified multipotent neural stem cell derivatives (hNSCs. This quantitative dataset (nearly 13,000 proteins and 60,000 phosphorylation sites provides unique molecular insights into pluripotency and neural lineage entry. Systems-level comparative analysis of proteins (e.g., transcription factors, epigenetic regulators, kinase families, phosphorylation sites, and numerous biological pathways allowed the identification of distinct signatures in pluripotent and multipotent cells. Furthermore, as predicted by the dataset, we functionally validated an autocrine/paracrine mechanism by demonstrating that the secreted protein midkine is a regulator of neural specification. This resource is freely available to the scientific community, including a searchable website, PluriProt.

  6. Sphingosine-1-Phosphate (S1P) Signaling in Neural Progenitors.

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    Callihan, Phillip; Alqinyah, Mohammed; Hooks, Shelley B

    2018-01-01

    Sphingosine-1-phosphate (S1P) and its receptors are important in nervous system development. Reliable in vitro human model systems are needed to further define specific roles for S1P signaling in neural development. We have described S1P-regulated signaling, survival, and differentiation in a human embryonic stem cell-derived neuroepithelial progenitor cell line (hNP1) that expresses functional S1P receptors. These cells can be further differentiated to a neuronal cell type and therefore represent a good model system to study the role of S1P signaling in human neural development. The following sections describe in detail the culture and differentiation of hNP1 cells and two assays to measure S1P signaling in these cells.

  7. Potential of Osteoblastic Cells Derived from Bone Marrow and Adipose Tissue Associated with a Polymer/Ceramic Composite to Repair Bone Tissue.

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    Freitas, Gileade P; Lopes, Helena B; Almeida, Adriana L G; Abuna, Rodrigo P F; Gimenes, Rossano; Souza, Lucas E B; Covas, Dimas T; Beloti, Marcio M; Rosa, Adalberto L

    2017-09-01

    One of the tissue engineering strategies to promote bone regeneration is the association of cells and biomaterials. In this context, the aim of this study was to evaluate if cell source, either from bone marrow or adipose tissue, affects bone repair induced by osteoblastic cells associated with a membrane of poly(vinylidene-trifluoroethylene)/barium titanate (PVDF-TrFE/BT). Mesenchymal stem cells (MSC) were isolated from rat bone marrow and adipose tissue and characterized by detection of several surface markers. Also, both cell populations were cultured under osteogenic conditions and it was observed that MSC from bone marrow were more osteogenic than MSC from adipose tissue. The bone repair was evaluated in rat calvarial defects implanted with PVDF-TrFE/BT membrane and locally injected with (1) osteoblastic cells differentiated from MSC from bone marrow, (2) osteoblastic cells differentiated from MSC from adipose tissue or (3) phosphate-buffered saline. Luciferase-expressing osteoblastic cells derived from bone marrow and adipose tissue were detected in bone defects after cell injection during 25 days without difference in luciferin signal between cells from both sources. Corroborating the in vitro findings, osteoblastic cells from bone marrow combined with the PVDF-TrFE/BT membrane increased the bone formation, whereas osteoblastic cells from adipose tissue did not enhance the bone repair induced by the membrane itself. Based on these findings, it is possible to conclude that, by combining a membrane with cells in this rat model, cell source matters and that bone marrow could be a more suitable source of cells for therapies to engineer bone.

  8. Initiation and Propagation of Vascular Calcification Is Regulated by a Concert of Platelet- and Smooth Muscle Cell-Derived Extracellular Vesicles

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    Leon J. Schurgers

    2018-04-01

    Full Text Available The ageing population continues to suffer from its primary killer, cardiovascular disease (CVD. Despite recent advances in interventional medicinal and surgical therapies towards the end of the 20th century, the epidemic of cardiovascular disease has not been halted. Yet, rather than receding globally, the burden of CVD has risen to become a top cause of morbidity and mortality worldwide. Most CVD arises from thrombotic rupture of an atherosclerotic plaque, the pathologic thickening of coronary and carotid artery segments and subsequent distal ischemia in heart or brain. In fact, one-fifth of deaths are directly attributable to thrombotic rupture of a vulnerable plaque. Atherosclerotic lesion formation is caused by a concert of interactions between circulating leukocytes and platelets, interacting with the endothelial barrier, signalling into the arterial wall by the release of cytokines and extracellular vesicles (EVs. Both platelet- and cell-derived EVs represent a novel mechanism of cellular communication, particularly by the transport and transfer of cargo and by reprogramming of the recipient cell. These interactions result in phenotypic switching of vascular smooth muscle cells (VSMCs causing migration and proliferation, and subsequent secretion of EVs. Loss of VSMCs attracts perivascular Mesenchymal Stem Cells (MSCs from the adventitia, which are a source of VSMCs and contribute to repair after vascular injury. However, continuous stress stimuli eventually switch phenotype of cells into osteochondrogenic VSMCs facilitating vascular calcification. Although Virchow’s triad is over 100 years old, it is a reality that is accurate today. It can be briefly summarised as changes in the composition of blood (platelet EVs, alterations in the vessel wall (VSMC phenotypic switching, MSC infiltration and EV release and disruption of blood flow (atherothrombosis. In this paper, we review the latest relevant advances in the identification of

  9. Distinguishing Petroleum (Crude Oil and Fuel) From Smoke Exposure within Populations Based on the Relative Blood Levels of Benzene, Toluene, Ethylbenzene, and Xylenes (BTEX), Styrene and 2,5-Dimethylfuran by Pattern Recognition Using Artificial Neural Networks.

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    Chambers, D M; Reese, C M; Thornburg, L G; Sanchez, E; Rafson, J P; Blount, B C; Ruhl, J R E; De Jesús, V R

    2018-01-02

    Studies of exposure to petroleum (crude oil/fuel) often involve monitoring benzene, toluene, ethylbenzene, xylenes (BTEX), and styrene (BTEXS) because of their toxicity and gas-phase prevalence, where exposure is typically by inhalation. However, BTEXS levels in the general U.S. population are primarily from exposure to tobacco smoke, where smokers have blood levels on average up to eight times higher than nonsmokers. This work describes a method using partition theory and artificial neural network (ANN) pattern recognition to classify exposure source based on relative BTEXS and 2,5-dimethylfuran blood levels. A method using surrogate signatures to train the ANN was validated by comparing blood levels among cigarette smokers from the National Health and Nutrition Examination Survey (NHANES) with BTEXS and 2,5-dimethylfuran signatures derived from the smoke of machine-smoked cigarettes. Classification agreement for an ANN model trained with relative VOC levels was up to 99.8% for nonsmokers and 100.0% for smokers. As such, because there is limited blood level data on individuals exposed to crude oil/fuel, only surrogate signatures derived from crude oil and fuel were used for training the ANN. For the 2007-2008 NHANES data, the ANN model assigned 7 out of 1998 specimens (0.35%) and for the 2013-2014 NHANES data 12 out of 2906 specimens (0.41%) to the crude oil/fuel signature category.

  10. Identification and Characterization of 293T Cell-Derived Exosomes by Profiling the Protein, mRNA and MicroRNA Components.

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    Jing Li

    Full Text Available Cell-derived exosomes are leading candidates for in vivo drug delivery carriers. In particular, exosomes derived from 293T cells are used most frequently, although exosome dosing has varied greatly among studies. Considering their biological origin, it is crucial to characterize the molecular composition of exosomes if large doses are to be administered in clinical settings. In this study, we present the first comprehensive analysis of the protein, messenger RNA and microRNA profiles of 293T cell-derived exosomes; then, we characterized these data using Gene Ontology annotation and Kyoto Encyclopedia for Genes and Genomes pathway analysis. Our study will provide the basis for the selection of 293T cell-derived exosome drug delivery systems. Profiling the exosomal signatures of 293T cells will lead to a better understanding of 293T exosome biology and will aid in the identification of any harmful factors in exosomes that could cause adverse clinical effects.

  11. Highly efficient methods to obtain homogeneous dorsal neural progenitor cells from human and mouse embryonic stem cells and induced pluripotent stem cells.

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    Zhang, Meixiang; Ngo, Justine; Pirozzi, Filomena; Sun, Ying-Pu; Wynshaw-Boris, Anthony

    2018-03-15

    significant differences in the variation of PAX6 and SOX1-positive NPCs between the two human pluripotent cell-derived methods; therefore, both methods are suitable for producing stable dorsal NPCs. When further differentiated into mature neurons, NPCs gave rise to a population of almost exclusively forebrain cortical neurons, confirming the dorsal fate commitment of the progenitors. The methods described in this study show improvements over previously published studies and are highly efficient at differentiating human and mouse pluripotent cell types into dorsal PAX6-positive NPCs and eventually into forebrain cortical neurons.

  12. Human neural progenitors express functional lysophospholipid receptors that regulate cell growth and morphology

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    Callihan Phillip

    2008-12-01

    Full Text Available Abstract Background Lysophospholipids regulate the morphology and growth of neurons, neural cell lines, and neural progenitors. A stable human neural progenitor cell line is not currently available in which to study the role of lysophospholipids in human neural development. We recently established a stable, adherent human embryonic stem cell-derived neuroepithelial (hES-NEP cell line which recapitulates morphological and phenotypic features of neural progenitor cells isolated from fetal tissue. The goal of this study was to determine if hES-NEP cells express functional lysophospholipid receptors, and if activation of these receptors mediates cellular responses critical for neural development. Results Our results demonstrate that Lysophosphatidic Acid (LPA and Sphingosine-1-phosphate (S1P receptors are functionally expressed in hES-NEP cells and are coupled to multiple cellular signaling pathways. We have shown that transcript levels for S1P1 receptor increased significantly in the transition from embryonic stem cell to hES-NEP. hES-NEP cells express LPA and S1P receptors coupled to Gi/o G-proteins that inhibit adenylyl cyclase and to Gq-like phospholipase C activity. LPA and S1P also induce p44/42 ERK MAP kinase phosphorylation in these cells and stimulate cell proliferation via Gi/o coupled receptors in an Epidermal Growth Factor Receptor (EGFR- and ERK-dependent pathway. In contrast, LPA and S1P stimulate transient cell rounding and aggregation that is independent of EGFR and ERK, but dependent on the Rho effector p160 ROCK. Conclusion Thus, lysophospholipids regulate neural progenitor growth and morphology through distinct mechanisms. These findings establish human ES cell-derived NEP cells as a model system for studying the role of lysophospholipids in neural progenitors.

  13. Cancer Cell-Derived Extracellular Vesicles Are Associated with Coagulopathy Causing Ischemic Stroke via Tissue Factor-Independent Way: The OASIS-CANCER Study.

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    Oh Young Bang

    Full Text Available Cancer and stroke, which are known to be associated with one another, are the most common causes of death in the elderly. However, the pathomechanisms that lead to stroke in cancer patients are not well known. Circulating extracellular vesicles (EVs play a role in cancer-associated thrombosis and tumor progression. Therefore, we hypothesized that cancer cell-derived EVs cause cancer-related coagulopathy resulting in ischemic stroke.Serum levels of D-dimer and EVs expressing markers for cancer cells (epithelial cell adhesion molecule [CD326], tissue factor (TF [CD142], endothelial cells (CD31+CD42b-, and platelets (CD62P were measured using flow cytometry in (a 155 patients with ischemic stroke and active cancer (116 - cancer-related, 39 - conventional stroke mechanisms, (b 25 patients with ischemic stroke without cancer, (c 32 cancer patients without stroke, and (d 101 healthy subjects.The levels of cancer cell-derived EVs correlated with the levels of D-dimer and TF+ EVs. The levels of cancer cell-derived EVs (CD326+ and CD326+CD142+ were higher in cancer-related stroke than in other groups (P<0.05 in all the cases. Path analysis showed that cancer cell-derived EVs are related to stroke via coagulopathy as measured by D-dimer levels. Poor correlation was observed between TF+ EV and D-dimer, and path analysis demonstrated that cancer cell-derived EVs may cause cancer-related coagulopathy independent of the levels of TF+ EVs.Our findings suggest that cancer cell-derived EVs mediate coagulopathy resulting in ischemic stroke via TF-independent mechanisms.

  14. Murine pluripotent stem cells derived scaffold-free cartilage grafts from a micro-cavitary hydrogel platform.

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    He, Pengfei; Fu, Jiayin; Wang, Dong-An

    2016-04-15

    By means of appropriate cell type and scaffold, tissue-engineering approaches aim to construct grafts for cartilage repair. Pluripotent stem cells especially induced pluripotent stem cells (iPSCs) are of promising cell candidates due to the pluripotent plasticity and abundant cell source. We explored three dimensional (3D) culture and chondrogenesis of murine iPSCs (miPSCs) on an alginate-based micro-cavity hydrogel (MCG) platform in pursuit of fabricating synthetic-scaffold-free cartilage grafts. Murine embryonic stem cells (mESCs) were employed in parallel as the control. Chondrogenesis was fulfilled using a consecutive protocol via mesoderm differentiation followed by chondrogenic differentiation; subsequently, miPSC and mESC-seeded constructs were further respectively cultured in chondrocyte culture (CC) medium. Alginate phase in the constructs was then removed to generate a graft only comprised of induced chondrocytic cells and cartilaginous extracellular matrix (ECMs). We found that from the mESC-seeded constructs, formation of intact grafts could be achieved in greater sizes with relatively fewer chondrocytic cells and abundant ECMs; from miPSC-seeded constructs, relatively smaller sized cartilaginous grafts could be formed by cells with chondrocytic phenotype wrapped by abundant and better assembled collagen type II. This study demonstrated successful creation of pluripotent stem cells-derived cartilage/chondroid graft from a 3D MCG interim platform. By the support of materials and methodologies established from this study, particularly given the autologous availability of iPSCs, engineered autologous cartilage engraftment may be potentially fulfilled without relying on the limited and invasive autologous chondrocytes acquisition. In this study, we explored chondrogenic differentiation of pluripotent stem cells on a 3D micro-cavitary hydrogel interim platform and creation of pluripotent stem cells-derived cartilage/chondroid graft via a consecutive

  15. Evaluation of amniotic mesenchymal cell derivatives on cytokine production in equine alveolar macrophages: an in vitro approach to lung inflammation.

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    Zucca, Enrica; Corsini, Emanuela; Galbiati, Valentina; Lange-Consiglio, Anna; Ferrucci, Francesco

    2016-09-20

    Data obtained in both animal models and clinical trials suggest that cell-based therapies represent a potential therapeutic strategy for lung repair and remodeling. Recently, new therapeutic approaches based on the use of stem cell derivatives (e.g., conditioned medium (CM) and microvesicles (MVs)) to regenerate tissues and improve their functions were proposed. The aim of this study was to investigate the immunomodulatory effects of equine amniotic mesenchymal cell derivatives on lipopolysaccharide (LPS)-induced cytokine production in equine alveolar macrophages, which may be beneficial in lung inflammatory disorders such as recurrent airway obstruction (RAO) in horses. RAO shares many features with human asthma, including an increased number of cells expressing mRNA for interleukin (IL)-4 and IL-5 and a decreased expression of IFN-γ in bronchoalveolar lavage fluid (BALF) of affected horses. The release of TNF-α, IL-6, and TGF-β1 at different time points (1, 24, 48, and 72 h) was measured in equine alveolar macrophages stimulated or not with LPS (10 and 100 ng/mL) in the presence or absence of 10 % CM or 50 × 10(6) MVs/mL. Cytokines were measured using commercially available ELISA kits. For multiple comparisons, analysis of variance was used with Tukey post-hoc test. Differences were considered significant at p ≤ 0.05. Significant modulatory effects of CM on LPS-induced TNF-α release at 24 h, and of both CM and MVs on TNF-α release at 48 h were observed. A trend toward a modulatory effect of both CM and MVs on the release of TGF-β and possibly IL-6 was visible over time. Results support the potential use of CM and MVs in lung regenerative medicine, especially in situations in which TGF-β may be detrimental, such as respiratory allergy. Further studies should evaluate the potential clinical applications of CM and MVs in equine lung diseases, such as RAO and other inflammatory disorders.

  16. Amelioration of hypertriglyceridemia with hypo-alpha-cholesterolemia in LPL deficient mice by hematopoietic cell-derived LPL.

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    Yinyuan Ding

    Full Text Available BACKGROUND: Macrophage-derived lipoprotein lipase (LPL has been shown uniformly to promote atherosclerotic lesion formation while the extent to which it affects plasma lipid and lipoprotein levels varies in wild-type and hypercholesterolemic mice. It is known that high levels of LPL in the bulk of adipose tissue and skeletal muscle would certainly mask the contribution of macrophage LPL to metabolism of plasma lipoprotein. Therefore, we chose LPL deficient (LPL⁻/⁻ mice with severe hypertriglyceridemia as an alternative model to assess the role of macrophage LPL in plasma lipoprotein metabolism via bone marrow transplant, through which LPL will be produced mainly by hematopoietic cell-derived macrophages. METHODS AND RESULTS: Hypertriglyceridemic LPL⁻/⁻ mice were lethally irradiated, then transplanted with bone marrow from wild-type (LPL⁺/⁺ or LPL⁻/⁻ mice, respectively. Sixteen weeks later, LPL⁺/⁺ →LPL⁻/⁻ mice displayed significant reduction in plasma levels of triglyceride and cholesterol (408±44.9 vs. 2.7±0.5×10³ and 82.9±7.1 vs. 229.1±30.6 mg/dl, p<0.05, respectively, while a 2.7-fold increase in plasma high density lipoprotein- cholesterol (p<0.01 was observed, compared with LPL⁻/⁻→LPL⁻/⁻ control mice. The clearance rate for the oral fat load test in LPL⁺/⁺ →LPL⁻/⁻ mice was faster than that in LPL⁻/⁻→LPL⁻/⁻ mice, but slower than that in wild-type mice. Liver triglyceride content in LPL⁺/⁺→LPL⁻/⁻ mice was also significantly increased, compared with LPL⁻/⁻→LPL⁻/⁻ mice (6.8±0.7 vs. 4.6±0.5 mg/g wet tissue, p<0.05, n = 6. However, no significant change was observed in the expression levels of genes involved in hepatic lipid metabolism between the two groups. CONCLUSIONS: Hematopoietic cell-derived LPL could efficiently ameliorate severe hypertriglyceridemia and hypo-alpha-cholesterolemia at the compensation of increased triglyceride content of liver in LPL

  17. A preliminary study for constructing a bioartificial liver device with induced pluripotent stem cell-derived hepatocytes

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    Iwamuro Masaya

    2012-12-01

    Full Text Available Abstract Background Bioartificial liver systems, designed to support patients with liver failure, are composed of bioreactors and functional hepatocytes. Immunological rejection of the embedded hepatocytes by the host immune system is a serious concern that crucially degrades the performance of the device. Induced pluripotent stem (iPS cells are considered a desirable source for bioartificial liver systems, because patient-derived iPS cells are free from immunological rejection. The purpose of this paper was to test the feasibility of a bioartificial liver system with iPS cell-derived hepatocyte-like cells. Methods Mouse iPS cells were differentiated into hepatocyte-like cells by a multi-step differentiation protocol via embryoid bodies and definitive endoderm. Differentiation of iPS cells was evaluated by morphology, PCR assay, and functional assays. iPS cell-derived hepatocyte-like cells were cultured in a bioreactor module with a pore size of 0.2 μm for 7 days. The amount of albumin secreted into the circulating medium was analyzed by ELISA. Additionally, after a 7-day culture in a bioreactor module, cells were observed by a scanning electron microscope. Results At the final stage of the differentiation program, iPS cells changed their morphology to a polygonal shape with two nucleoli and enriched cytoplasmic granules. Transmission electron microscope analysis revealed their polygonal shape, glycogen deposition in the cytoplasm, microvilli on their surfaces, and a duct-like arrangement. PCR analysis showed increased expression of albumin mRNA over the course of the differentiation program. Albumin and urea production was also observed. iPS-Heps culture in bioreactor modules showed the accumulation of albumin in the medium for up to 7 days. Scanning electron microscopy revealed the attachment of cell clusters to the hollow fibers of the module. These results indicated that iPS cells were differentiated into hepatocyte-like cells after culture

  18. Neural Darwinism and consciousness.

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    Seth, Anil K; Baars, Bernard J

    2005-03-01

    Neural Darwinism (ND) is a large scale selectionist theory of brain development and function that has been hypothesized to relate to consciousness. According to ND, consciousness is entailed by reentrant interactions among neuronal populations in the thalamocortical system (the 'dynamic core'). These interactions, which permit high-order discriminations among possible core states, confer selective advantages on organisms possessing them by linking current perceptual events to a past history of value-dependent learning. Here, we assess the consistency of ND with 16 widely recognized properties of consciousness, both physiological (for example, consciousness is associated with widespread, relatively fast, low amplitude interactions in the thalamocortical system), and phenomenal (for example, consciousness involves the existence of a private flow of events available only to the experiencing subject). While no theory accounts fully for all of these properties at present, we find that ND and its recent extensions fare well.

  19. Chimera States in Neural Oscillators

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    Bahar, Sonya; Glaze, Tera

    2014-03-01

    Chimera states have recently been explored both theoretically and experimentally, in various coupled nonlinear oscillators, ranging from phase-oscillator models to coupled chemical reactions. In a chimera state, both coherent and incoherent (or synchronized and desynchronized) states occur simultaneously in populations of identical oscillators. We investigate chimera behavior in a population of neural oscillators using the Huber-Braun model, a Hodgkin-Huxley-like model originally developed to characterize the temperature-dependent bursting behavior of mammalian cold receptors. One population of neurons is allowed to synchronize, with each neuron receiving input from all the others in its group (global within-group coupling). Subsequently, a second population of identical neurons is placed under an identical global within-group coupling, and the two populations are also coupled to each other (between-group coupling). For certain values of the coupling constants, the neurons in the two populations exhibit radically different synchronization behavior. We will discuss the range of chimera activity in the model, and discuss its implications for actual neural activity, such as unihemispheric sleep.

  20. Variation in primary and culture-expanded cells derived from connective tissue progenitors in human bone marrow space, bone trabecular surface and adipose tissue.

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    Qadan, Maha A; Piuzzi, Nicolas S; Boehm, Cynthia; Bova, Wesley; Moos, Malcolm; Midura, Ronald J; Hascall, Vincent C; Malcuit, Christopher; Muschler, George F

    2018-03-01

    Connective tissue progenitors (CTPs) embody the heterogeneous stem and progenitor cell populations present in native tissue. CTPs are essential to the formation and remodeling of connective tissue and represent key targets for tissue-engineering and cell-based therapies. To better understand and characterize CTPs, we aimed to compare the (i) concentration and prevalence, (ii) early in vitro biological behavior and (iii) expression of surface-markers and transcription factors among cells derived from marrow space (MS), trabecular surface (TS), and adipose tissues (AT). Cancellous-bone and subcutaneous-adipose tissues were collected from 8 patients. Cells were isolated and cultured. Colony formation was assayed using Colonyze software based on ASTM standards. Cell concentration ([Cell]), CTP concentration ([CTP]) and CTP prevalence (P CTP ) were determined. Attributes of culture-expanded cells were compared based on (i) effective proliferation rate and (ii) expression of surface-markers CD73, CD90, CD105, SSEA-4, SSEA-3, SSEA-1/CD15, Cripto-1, E-Cadherin/CD324, Ep-CAM/CD326, CD146, hyaluronan and transcription factors Oct3/4, Sox-2 and Nanog using flow cytometry. Mean [Cell], [CTP] and P CTP were significantly different between MS and TS samples (P = 0.03, P = 0.008 and P= 0.0003), respectively. AT-derived cells generated the highest mean total cell yield at day 6 of culture-4-fold greater than TS and more than 40-fold greater than MS per million cells plated. TS colonies grew with higher mean density than MS colonies (290 ± 11 versus 150 ± 11 cell per mm 2 ; P = 0.0002). Expression of classical-mesenchymal stromal cell (MSC) markers was consistently recorded (>95%) from all tissue sources, whereas all the other markers were highly variable. The prevalence and biological potential of CTPs are different between patients and tissue sources and lack variation in classical MSC markers. Other markers are more likely to discriminate differences

  1. Phenotypic variability in LQT3 human induced pluripotent stem cell-derived cardiomyocytes and their response to antiarrhythmic pharmacologic therapy: An in silico approach.

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    Paci, Michelangelo; Passini, Elisa; Severi, Stefano; Hyttinen, Jari; Rodriguez, Blanca

    2017-11-01

    Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are in vitro models with the clear advantages of their human origin and suitability for human disease investigations. However, limitations include their incomplete characterization and variability reported in different cell lines and laboratories. The purpose of this study was to investigate in silico ionic mechanisms potentially explaining the phenotypic variability of hiPSC-CMs in long QT syndrome type 3 (LQT3) and their response to antiarrhythmic drugs. Populations of in silico hiPSC-CM models were constructed and calibrated for control (n = 1,463 models) and LQT3 caused by I NaL channelopathy (n = 1,401 models), using experimental recordings for late sodium current (I NaL ) and action potentials (APs). Antiarrhythmic drug therapy was evaluated by simulating mexiletine and ranolazine multichannel effects. As in experiments, LQT3 hiPSC-CMs yield prolonged action potential duration at 90% repolarization (APD 90 ) (+34.3% than controls) and large electrophysiological variability. LQT3 hiPSC-CMs with symptomatic APs showed overexpression of I CaL , I K1 , and I NaL , underexpression of I Kr , and increased sensitivity to both drugs compared to asymptomatic LQT3 models. Simulations showed that both mexiletine and ranolazine corrected APD prolongation in the LQT3 population but also highlighted differences in drug response. Mexiletine stops spontaneous APs in more LQT3 hiPSC-CMs models than ranolazine (784/1,401 vs 53/1,401) due to its stronger action on I Na . In silico simulations demonstrate our ability to recapitulate variability in LQT3 and control hiPSC-CM phenotypes, and the ability of mexiletine and ranolazine to reduce APD prolongation, in agreement with experiments. The in silico models also identify potential ionic mechanisms of phenotypic variability in LQT3 hiPSC-CMs, explaining APD prolongation in symptomatic vs asymptomatic LQT3 hiPSC-CMs. Copyright © 2017 The Authors. Published by

  2. Human Induced Pluripotent Stem Cell-Derived Macrophages Share Ontogeny with MYB-Independent Tissue-Resident Macrophages

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    Julian Buchrieser

    2017-02-01

    Full Text Available Tissue-resident macrophages, such as microglia, Kupffer cells, and Langerhans cells, derive from Myb-independent yolk sac (YS progenitors generated before the emergence of hematopoietic stem cells (HSCs. Myb-independent YS-derived resident macrophages self-renew locally, independently of circulating monocytes and HSCs. In contrast, adult blood monocytes, as well as infiltrating, gut, and dermal macrophages, derive from Myb-dependent HSCs. These findings are derived from the mouse, using gene knockouts and lineage tracing, but their applicability to human development has not been formally demonstrated. Here, we use human induced pluripotent stem cells (iPSCs as a tool to model human hematopoietic development. By using a CRISPR-Cas9 knockout strategy, we show that human iPSC-derived monocytes/macrophages develop in an MYB-independent, RUNX1-, and SPI1 (PU.1-dependent fashion. This result makes human iPSC-derived macrophages developmentally related to and a good model for MYB-independent tissue-resident macrophages, such as alveolar and kidney macrophages, microglia, Kupffer cells, and Langerhans cells.

  3. Feasibility of autologous bone marrow mesenchymal stem cell-derived extracellular matrix scaffold for cartilage tissue engineering.

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    Tang, Cheng; Xu, Yan; Jin, Chengzhe; Min, Byoung-Hyun; Li, Zhiyong; Pei, Xuan; Wang, Liming

    2013-12-01

    Extracellular matrix (ECM) materials are widely used in cartilage tissue engineering. However, the current ECM materials are unsatisfactory for clinical practice as most of them are derived from allogenous or xenogenous tissue. This study was designed to develop a novel autologous ECM scaffold for cartilage tissue engineering. The autologous bone marrow mesenchymal stem cell-derived ECM (aBMSC-dECM) membrane was collected and fabricated into a three-dimensional porous scaffold via cross-linking and freeze-drying techniques. Articular chondrocytes were seeded into the aBMSC-dECM scaffold and atelocollagen scaffold, respectively. An in vitro culture and an in vivo implantation in nude mice model were performed to evaluate the influence on engineered cartilage. The current results showed that the aBMSC-dECM scaffold had a good microstructure and biocompatibility. After 4 weeks in vitro culture, the engineered cartilage in the aBMSC-dECM scaffold group formed thicker cartilage tissue with more homogeneous structure and higher expressions of cartilaginous gene and protein compared with the atelocollagen scaffold group. Furthermore, the engineered cartilage based on the aBMSC-dECM scaffold showed better cartilage formation in terms of volume and homogeneity, cartilage matrix content, and compressive modulus after 3 weeks in vivo implantation. These results indicated that the aBMSC-dECM scaffold could be a successful novel candidate scaffold for cartilage tissue engineering. © 2013 Wiley Periodicals, Inc. and International Center for Artificial Organs and Transplantation.

  4. Characterization of progenitor cells derived from torn human rotator cuff tendons by gene expression patterns of chondrogenesis, osteogenesis, and adipogenesis.

    Science.gov (United States)

    Nagura, Issei; Kokubu, Takeshi; Mifune, Yutaka; Inui, Atsuyuki; Takase, Fumiaki; Ueda, Yasuhiro; Kataoka, Takeshi; Kurosaka, Masahiro

    2016-03-31

    It is important to regenerate the tendon-to-bone interface after rotator cuff repair to prevent re-tears. The cells from torn human rotator cuff were targeted, and their capacity for multilineage differentiation was investigated. The edges of the rotator cuff were harvested during arthroscopic rotator cuff repair from nine patients, minced into pieces, and cultured on dishes. Adherent cells were cultured, phenotypically characterized. Then expandability, differentiation potential and gene expression were analyzed. Flow cytometry revealed that the mesenchymal stem cells (MSC)-related markers CD29, CD44, CD105, and CD166 were positive. However, CD14, CD34, and CD45 were negative. On RT-PCR analyses, the cells showed osteogenic, adipogenic, and chondrogenic potential after 3 weeks of culture under the respective differentiation conditions. In addition, SOX9, type II collagen, and type X collagen expression patterns during chondrogenesis were similar to those of endochondral ossification at the enthesis. The cells derived from torn human rotator cuff are multipotent mesenchymal stem cells with the ability to undergo multilineage differentiation, suggesting that MSCs form this tissue could be regenerative capacity for potential self-repair.

  5. Successful immortalization of mesenchymal progenitor cells derived from human placenta and the differentiation abilities of immortalized cells

    International Nuclear Information System (INIS)

    Zhang Xiaohong; Soda, Yasushi; Takahashi, Kenji; Bai, Yuansong; Mitsuru, Ayako; Igura, Koichi; Satoh, Hitoshi; Yamaguchi, Satoru; Tani, Kenzaburo; Tojo, Arinobu; Takahashi, Tsuneo A.

    2006-01-01

    We reported previously that mesenchymal progenitor cells derived from chorionic villi of the human placenta could differentiate into osteoblasts, adipocytes, and chondrocytes under proper induction conditions and that these cells should be useful for allogeneic regenerative medicine, including cartilage tissue engineering. However, similar to human mesenchymal stem cells (hMSCs), though these placental cells can be isolated easily, they are difficult to study in detail because of their limited life span in vitro. To overcome this problem, we attempted to prolong the life span of human placenta-derived mesenchymal cells (hPDMCs) by modifying hTERT and Bmi-1, and investigated whether these modified hPDMCs retained their differentiation capability and multipotency. Our results indicated that the combination of hTERT and Bmi-1 was highly efficient in prolonging the life span of hPDMCs with differentiation capability to osteogenic, adipogenic, and chondrogenic cells in vitro. Clonal cell lines with directional differentiation ability were established from the immortalized parental hPDMC/hTERT + Bmi-1. Interestingly, hPDMC/Bmi-1 showed extended proliferation after long-term growth arrest and telomerase was activated in the immortal hPDMC/Bmi-1 cells. However, the differentiation potential was lost in these cells. This study reports a method to extend the life span of hPDMCs with hTERT and Bmi-1 that should become a useful tool for the study of mesenchymal stem cells

  6. Long-term Culture of Human iPS Cell-derived Telencephalic Neuron Aggregates on Collagen Gel.

    Science.gov (United States)

    Oyama, Hiroshi; Takahashi, Koji; Tanaka, Yoshikazu; Takemoto, Hiroshi; Haga, Hisashi

    2018-01-01

    It takes several months to form the 3-dimensional morphology of the human embryonic brain. Therefore, establishing a long-term culture method for neuronal tissues derived from human induced pluripotent stem (iPS) cells is very important for studying human brain development. However, it is difficult to keep primary neurons alive for more than 3 weeks in culture. Moreover, long-term adherent culture to maintain the morphology of telencephalic neuron aggregates induced from human iPS cells is also difficult. Although collagen gel has been widely used to support long-term culture of cells, it is not clear whether human iPS cell-derived neuron aggregates can be cultured for long periods on this substrate. In the present study, we differentiated human iPS cells to telencephalic neuron aggregates and examined long-term culture of these aggregates on collagen gel. The results indicated that these aggregates could be cultured for over 3 months by adhering tightly onto collagen gel. Furthermore, telencephalic neuronal precursors within these aggregates matured over time and formed layered structures. Thus, long-term culture of telencephalic neuron aggregates derived from human iPS cells on collagen gel would be useful for studying human cerebral cortex development.Key words: Induced pluripotent stem cell, forebrain neuron, collagen gel, long-term culture.

  7. Cytotoxicity of Silver Nanoparticles in Human Embryonic Stem Cell-Derived Fibroblasts and an L-929 Cell Line

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    Hui Peng

    2012-01-01

    Full Text Available Consensus about the toxicity of silver nanoparticles (Ag-NPs has not been reached, even though extensive attention has been paid to this issue. This confusion may be due to physicochemical factors of Ag-NPs and the cell model used for biological safety evaluation. In the present study, human embryonic stem cell-derived fibroblasts (EBFs, which have been considered a closer representative of the in vivo response, were used as a novel cell model to assess the cytotoxicity of Ag-NPs (~20 nm and ~100 nm in comparison with L-929 fibroblast cell line. Cell proliferation, cell cycle, apoptosis, p53 expression, and cellular uptake were examined. Results showed that Ag-NPs presented higher cytotoxicity to EBF than to L-929. EBF demonstrated a stronger capacity to ingest Ag-NPs, a higher G2/M arrest, and more upgraduated p53 expression after exposed to Ag-NPs for 48 h when compared with L-929. It could be concluded that EBF exhibited a more sensitive response to Ag-NPs compared with L-929 cells, indicating that EBF may be a valid candidate for cytotoxicity screening assays of nanoparticles.

  8. Vildagliptin reduces plasma stromal cell-derived factor-1α in patients with type 2 diabetes compared with glimepiride.

    Science.gov (United States)

    Park, Kyeong Seon; Kwak, SooHeon; Cho, Young Min; Park, Kyong Soo; Jang, Hak C; Kim, Seong Yeon; Jung, Hye Seung

    2017-03-01

    Dipeptidyl peptidase-4 inhibitors might have pleiotropic protective effects on cardiovascular disease (CVD), in contrast to sulfonylureas. Therefore, we compared various CVD risk factors between vildagliptin and glimepiride. We carried out a randomized, prospective and crossover trial. A total of 16 patients with type 2 diabetes whose glycated hemoglobin was >7% were randomized to add vildagliptin or glimepiride. After 12-week treatment, each drug was replaced with the other for another 12 weeks. Before and after each treatment, glucose homeostasis and CVD risk factors were assessed, and the continuous glucose monitoring system was applied to calculate glycemic variability. The mean age of the participants was 60 years, 31% were men, body mass index 25.5 kg/m 2 and HbA1c 8.41%. Both vildagliptin and glimepiride significantly decreased glycated hemoglobin and glycemic variability indices. Despite the improved glucose homeostasis, favorable change of CVD markers was not prominent in both the arms, along with significant weight gain. Only plasma stromal cell-derived factor (SDF)-1α decreased by 30% in the vildagliptin arm. According to regression analyses, the reduction of SDF-1α was independently associated with vildagliptin usage and serum interleukin-6 changes, but white blood cells were not related with the SDF-1α changes. Compared with glimepiride, vildagliptin arrestingly decreased plasma SDF-1α, and its clinical implications should be further investigated. © 2016 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.

  9. Interleukin-1β-induced autophagy-related gene 5 regulates proliferation of embryonic stem cell-derived odontoblastic cells.

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    Nobuaki Ozeki

    Full Text Available We previously established a method for the differentiation of induced pluripotent stem cells and embryonic stem cells into α2 integrin-positive odontoblast-like cells. We also reported that Wnt5 in response to interleukin (IL-1β induces matrix metalloproteinase (MMP-3-regulated cell proliferation in these cells. Our findings suggest that MMP-3 plays a potentially unique physiological role in the generation of odontoblast-like cells under an inflammatory state. Here, we examined whether up-regulation of autophagy-related gene (Atg 5 by IL-1β was mediated by Wnt5 signaling, thus leading to increased proliferation of odontoblast-like cells. IL-1β increased the mRNA and protein levels of Atg5, microtubule-associated protein 1 light chain (LC3, a mammalian homolog of yeast Atg8 and Atg12. Treatment with siRNAs against Atg5, but not LC3 and Atg12, suppressed the IL-1β-induced increase in MMP-3 expression and cell proliferation. Our siRNA analyses combined with western blot analysis revealed a unique sequential cascade involving Atg5, Wnt5a and MMP-3, which resulted in the potent increase in odontoblastic cell proliferation. These results demonstrate the unique involvement of Atg5 in IL-1β-induced proliferation of embryonic stem cell-derived odontoblast-like cells.

  10. Patient-specific cardiovascular progenitor cells derived from integration-free induced pluripotent stem cells for vascular tissue regeneration.

    Science.gov (United States)

    Hu, Jiang; Wang, Yongyu; Jiao, Jiao; Liu, Zhongning; Zhao, Chao; Zhou, Zhou; Zhang, Zhanpeng; Forde, Kaitlynn; Wang, Lunchang; Wang, Jiangang; Baylink, David J; Zhang, Xiao-Bing; Gao, Shaorong; Yang, Bo; Chen, Y Eugene; Ma, Peter X

    2015-12-01

    Tissue-engineered blood vessels (TEBVs) are promising in regenerating a live vascular replacement. However, the vascular cell source is limited, and it is crucial to develop a scaffold that accommodates new type of vascular progenitor cells and facilitates in vivo lineage specification of the cells into functional vascular smooth muscle cells (VSMCs) to regenerate vascular tissue. In the present study, integration-free human induced pluripotent stem cells (hiPSCs) were established from patient peripheral blood mononuclear cells through episomal vector nucleofection of reprogramming factors. The established hiPSCs were then induced into mesoderm-originated cardiovascular progenitor cells (CVPCs) with a highly efficient directed lineage specification method. The derived CVPCs were demonstrated to be able to differentiate into functional VSMCs. Subcutaneous implantation of CVPCs seeded on macroporous nanofibrous poly(l-lactide) scaffolds led to in vivo VSMC lineage specification and matrix deposition inside the scaffolds. In summary, we established integration-free patient-specific hiPSCs from peripheral blood mononuclear cells, derived CVPCs through directed lineage specification, and developed an advanced scaffold for these progenitor cells to further differentiate in vivo into VSMCs and regenerate vascular tissue in a subcutaneous implantation model. This study has established an efficient patient-specific approach towards in vivo regeneration of vascular tissue. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Embryonic Stem Cell-Derived Cardiomyocyte Heterogeneity and the Isolation of Immature and Committed Cells for Cardiac Remodeling and Regeneration

    Directory of Open Access Journals (Sweden)

    Kenneth R. Boheler

    2011-01-01

    Full Text Available Pluripotent stem cells represent one promising source for cell replacement therapy in heart, but differentiating embryonic stem cell-derived cardiomyocytes (ESC-CMs are highly heterogeneous and show a variety of maturation states. In this study, we employed an ESC clonal line that contains a cardiac-restricted ncx1 promoter-driven puromycin resistance cassette together with a mass culture system to isolate ESC-CMs that display traits characteristic of very immature CMs. The cells display properties of proliferation, CM-restricted markers, reduced mitochondrial mass, and hypoxia-resistance. Following transplantation into rodent hearts, bioluminescence imaging revealed that immature cells, but not more mature CMs, survived for at least one month following injection. These data and comparisons with more mature cells lead us to conclude that immature hypoxia resistant ESC-CMs can be isolated in mass in vitro and, following injection into heart, form grafts that may mediate long-term recovery of global and regional myocardial contractile function following infarction.

  12. B cell-derived transforming growth factor-β1 expression limits the induction phase of autoimmune neuroinflammation.

    Science.gov (United States)

    Bjarnadóttir, Kristbjörg; Benkhoucha, Mahdia; Merkler, Doron; Weber, Martin S; Payne, Natalie L; Bernard, Claude C A; Molnarfi, Nicolas; Lalive, Patrice H

    2016-10-06

    Studies in experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS), have shown that regulatory B cells modulate the course of the disease via the production of suppressive cytokines. While data indicate a role for transforming growth factor (TGF)-β1 expression in regulatory B cell functions, this mechanism has not yet been tested in autoimmune neuroinflammation. Transgenic mice deficient for TGF-β1 expression in B cells (B-TGF-β1 -/- ) were tested in EAE induced by recombinant mouse myelin oligodendrocyte glycoprotein (rmMOG). In this model, B-TGF-β1 -/- mice showed an earlier onset of neurologic impairment compared to their littermate controls. Exacerbated EAE susceptibility in B-TGF-β1 -/- mice was associated with augmented CNS T helper (Th)1/17 responses. Moreover, selective B cell TGF-β1-deficiency increased the frequencies and activation of myeloid dendritic cells, potent professional antigen-presenting cells (APCs), suggesting that B cell-derived TGF-β1 can constrain Th1/17 responses through inhibition of APC activity. Collectively our data suggest that B cells can down-regulate the function of APCs, and in turn encephalitogenic Th1/17 responses, via TGF-β1, findings that may be relevant to B cell-targeted therapies.

  13. Transplanted Human Stem Cell-Derived Interneuron Precursors Mitigate Mouse Bladder Dysfunction and Central Neuropathic Pain after Spinal Cord Injury.

    Science.gov (United States)

    Fandel, Thomas M; Trivedi, Alpa; Nicholas, Cory R; Zhang, Haoqian; Chen, Jiadong; Martinez, Aida F; Noble-Haeusslein, Linda J; Kriegstein, Arnold R

    2016-10-06

    Neuropathic pain and bladder dysfunction represent significant quality-of-life issues for many spinal cord injury patients. Loss of GABAergic tone in the injured spinal cord may contribute to the emergence of these symptoms. Previous studies have shown that transplantation of rodent inhibitory interneuron precursors from the medial ganglionic eminence (MGE) enhances GABAergic signaling in the brain and spinal cord. Here we look at whether transplanted MGE-like cells derived from human embryonic stem cells (hESC-MGEs) can mitigate the pathological effects of spinal cord injury. We find that 6 months after transplantation into injured mouse spinal cords, hESC-MGEs differentiate into GABAergic neuron subtypes and receive synaptic inputs, suggesting functional integration into host spinal cord. Moreover, the transplanted animals show improved bladder function and mitigation of pain-related symptoms. Our results therefore suggest that this approach may be a valuable strategy for ameliorating the adverse effects of spinal cord injury. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Three-Dimensional Culture of Human Embryonic Stem Cell Derived Hepatic Endoderm and Its Role in Bioartificial Liver Construction

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    Ruchi Sharma

    2010-01-01

    Full Text Available The liver carries out a range of functions essential for bodily homeostasis. The impairment of liver functions has serious implications and is responsible for high rates of patient morbidity and mortality. Presently, liver transplantation remains the only effective treatment, but donor availability is a major limitation. Therefore, artificial and bioartificial liver devices have been developed to bridge patients to liver transplantation. Existing support devices improve hepatic encephalopathy to a certain extent; however their usage is associated with side effects. The major hindrance in the development of bioartificial liver devices and cellular therapies is the limited availability of human hepatocytes. Moreover, primary hepatocytes are difficult to maintain and lose hepatic identity and function over time even with sophisticated tissue culture media. To overcome this limitation, renewable cell sources are being explored. Human embryonic stem cells are one such cellular resource and have been shown to generate a reliable and reproducible supply of human hepatic endoderm. Therefore, the use of human embryonic stem cell-derived hepatic endoderm in combination with tissue engineering has the potential to pave the way for the development of novel bioartificial liver devices and predictive drug toxicity assays.

  15. Stem cell-derived vasculature: A potent and multidimensional technology for basic research, disease modeling, and tissue engineering.

    Science.gov (United States)

    Lowenthal, Justin; Gerecht, Sharon

    2016-05-06

    Proper blood vessel networks are necessary for constructing and re-constructing tissues, promoting wound healing, and delivering metabolic necessities throughout the body. Conversely, an understanding of vascular dysfunction has provided insight into the pathogenesis and progression of diseases both common and rare. Recent advances in stem cell-based regenerative medicine - including advances in stem cell technologies and related progress in bioscaffold design and complex tissue engineering - have allowed rapid advances in the field of vascular biology, leading in turn to more advanced modeling of vascular pathophysiology and improved engineering of vascularized tissue constructs. In this review we examine recent advances in the field of stem cell-derived vasculature, providing an overview of stem cell technologies as a source for vascular cell types and then focusing on their use in three primary areas: studies of vascular development and angiogenesis, improved disease modeling, and the engineering of vascularized constructs for tissue-level modeling and cell-based therapies. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Economic 3D-printing approach for transplantation of human stem cell-derived β-like cells.

    Science.gov (United States)

    Song, Jiwon; Millman, Jeffrey R

    2016-12-01

    Transplantation of human pluripotent stem cells (hPSC) differentiated into insulin-producing β cells is a regenerative medicine approach being investigated for diabetes cell replacement therapy. This report presents a multifaceted transplantation strategy that combines differentiation into stem cell-derived β (SC-β) cells with 3D printing. By modulating the parameters of a low-cost 3D printer, we created a macroporous device composed of polylactic acid (PLA) that houses SC-β cell clusters within a degradable fibrin gel. Using finite element modeling of cellular oxygen diffusion-consumption and an in vitro culture system that allows for culture of devices at physiological oxygen levels, we identified cluster sizes that avoid severe hypoxia within 3D-printed devices and developed a microwell-based technique for resizing clusters within this range. Upon transplantation into mice, SC-β cell-embedded 3D-printed devices function for 12 weeks, are retrievable, and maintain structural integrity. Here, we demonstrate a novel 3D-printing approach that advances the use of differentiated hPSC for regenerative medicine applications and serves as a platform for future transplantation strategies.

  17. Alleviation of lipopolysaccharide/d-galactosamine-induced liver injury in leukocyte cell-derived chemotaxin 2 deficient mice

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    Akinori Okumura

    2017-12-01

    Full Text Available Leukocyte cell-derived chemotaxin 2 (LECT2 is a secreted pleiotropic protein that is mainly produced by the liver. We have previously shown that LECT2 plays an important role in the pathogenesis of inflammatory liver diseases. Lipopolysaccharide/d-galactosamine (LPS/d-GalN-induced acute liver injury is a known animal model of fulminant hepatic failure. Here we found that this hepatic injury was alleviated in LECT2-deficient mice. The levels of TNF-α and IFN-γ, which mediate this hepatitis, had significantly decreased in these mice, with the decrease in IFN-γ production notably greater than that in TNF-α. We therefore analyzed IFN-γ-producing cells in liver mononuclear cells. Flow cytometric analysis showed significantly reduced IFN-γ production in hepatic NK and NKT cells in LECT2-deficient mice compared with in wild-type mice. We also demonstrated a decrease in IFN-γ production in LECT2-deficient mice after systemic administration of recombinant IL-12, which is known to induce IFN-γ in NK and NKT cells. These results indicate that a decrease of IFN-γ production in NK and NKT cells was involved in the alleviation of LPS/d-GalN-induced liver injury in LECT2-deficient mice.

  18. BRICHOS domain-containing leukocyte cell-derived chemotaxin 1-like cDNA from disk abalone Haliotis discus discus.

    Science.gov (United States)

    Kim, Yucheol; De Zoysa, Mahanama; Lee, Youngdeuk; Whang, Ilson; Lee, Jehee

    2010-11-01

    A BRICHOS domain-containing leukocyte cell-derived chemotaxin 1-like cDNA was cloned from the disk abalone (Haliotis discus discus) and designated as AbLECT-1. A full-length (705 bp) of AbLECT-1 cDNA was composed of a 576 bp open reading frame that translates into a putative peptide of 192 amino acids. Deduced amino acid sequence of AbLECT-1 had 15.5- and 27.8% identity and similarity to human LECT-1, respectively. Quantitative real-time PCR analysis results showed that the mRNA of AbLECT-1 was constitutively expressed in abalone hemocytes, gills, mantle, muscle, digestive tract and hepatopancreas in a tissue-specific manner. Moreover, the AbLECT-1 transcription level was induced in hemocytes after challenge with Vibrio alginolyticus, Vibrio parahemolyticus, and Listeria monocytogenes suggesting that it may be involved in immune response reactions in abalone. Copyright 2010 Elsevier Ltd. All rights reserved.

  19. Homozygous mutation of focal adhesion kinase in embryonic stem cell derived neurons: normal electrophysiological and morphological properties in vitro

    Directory of Open Access Journals (Sweden)

    Komiyama NH

    2006-06-01

    Full Text Available Abstract Background Genetically manipulated embryonic stem (ES cell derived neurons (ESNs provide a powerful system with which to study the consequences of gene manipulation in mature, synaptically connected neurons in vitro. Here we report a study of focal adhesion kinase (FAK, which has been implicated in synapse formation and regulation of ion channels, using the ESN system to circumvent the embryonic lethality of homozygous FAK mutant mice. Results Mouse ES cells carrying homozygous null mutations (FAK-/- were generated and differentiated in vitro into neurons. FAK-/- ESNs extended axons and dendrites and formed morphologically and electrophysiologically intact synapses. A detailed study of NMDA receptor gated currents and voltage sensitive calcium currents revealed no difference in their magnitude, or modulation by tyrosine kinases. Conclusion FAK does not have an obligatory role in neuronal differentiation, synapse formation or the expression of NMDA receptor or voltage-gated calcium currents under the conditions used in this study. The use of genetically modified ESNs has great potential for rapidly and effectively examining the consequences of neuronal gene manipulation and is complementary to mouse studies.

  20. Magnetic tagging of cell-derived microparticles: new prospects for imaging and manipulation of these mediators of biological information.

    Science.gov (United States)

    Vats, Nidhi; Wilhelm, Claire; Rautou, Pierre-Emmanuel; Poirier-Quinot, Marie; Péchoux, Christine; Devue, Cécile; Boulanger, Chantal M; Gazeau, Florence

    2010-07-01

    Submicron membrane fragments termed microparticles (MPs), which are released by apoptotic or activated cells, are newly considered as vectors of biological information and actors of pathology development. We propose the tagging of MPs with magnetic nanoparticles as a new approach allowing imaging, manipulation and targeting of cell-derived MPs. MPs generated in vitro from human endothelial cells or isolated from atherosclerotic plaques were labeled using citrate-coated 8 nm iron-oxide nanoparticles. MPs were tagged with magnetic nanoparticles on their surface and detected as Annexin-V positive by flow cytometry. Labeled MPs could be mobilized, isolated and manipulated at a distance in a magnetic field gradient. Magnetic mobility of labeled MPs was quantified by micromagnetophoresis. Interactions of labeled MPs with endothelial cells could be triggered and modulated by magnetic guidance. Nanoparticles served as tracers at different scales: at the subcellular level by electron microscopy, at the cellular level by histology and at the macroscopic level by MRI. Magnetic labeling of biogenic MPs opens new prospects for noninvasive monitoring and distal manipulations of these biological effectors.

  1. Increased circulating cell-derived microparticle count is associated with recurrent implantation failure after IVF and embryo transfer.

    Science.gov (United States)

    Martínez-Zamora, M Angeles; Tàssies, Dolors; Reverter, Juan Carlos; Creus, Montserrat; Casals, Gemma; Cívico, Salvadora; Carmona, Francisco; Balasch, Juan

    2016-08-01

    Cell-derived microparticles (cMPs) are small membrane vesicles that are released from many different cell types in response to cellular activation or apoptosis. Elevated cMP counts have been found in almost all thrombotic diseases and pregnancy wastage, such as recurrent spontaneous abortion and in a number of conditions associated with inflammation, cellular activation and angiogenesis. cMP count was investigated in patients experiencing unexplained recurrent implantation failure (RIF). The study group was composed of 30 women diagnosed with RIF (RIF group). The first control group (IVF group) (n = 30) comprised patients undergoing a first successful IVF cycle. The second control group (FER group) included 30 healthy women who had at least one child born at term and no history of infertility or obstetric complications. cMP count was significantly higher in the RIF group compared with the IVF and FER groups (P < 0.05 and P < 0.01, respectively) (RIF group: 15.8 ± 6.2 nM phosphatidylserine equivalent [PS eq]; IVF group: 10.9 ± 5.3 nM PS eq; FER group: 9.6 ± 4.0 nM PS eq). No statistical difference was found in cMP count between the IVF and FER groups. Increased cMP count is, therefore, associated with RIF after IVF and embryo transfer. Copyright © 2016. Published by Elsevier Ltd.

  2. Morphological neural networks

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    Ritter, G.X.; Sussner, P. [Univ. of Florida, Gainesville, FL (United States)

    1996-12-31

    The theory of artificial neural networks has been successfully applied to a wide variety of pattern recognition problems. In this theory, the first step in computing the next state of a neuron or in performing the next layer neural network computation involves the linear operation of multiplying neural values by their synaptic strengths and adding the results. Thresholding usually follows the linear operation in order to provide for nonlinearity of the network. In this paper we introduce a novel class of neural networks, called morphological neural networks, in which the operations of multiplication and addition are replaced by addition and maximum (or minimum), respectively. By taking the maximum (or minimum) of sums instead of the sum of products, morphological network computation is nonlinear before thresholding. As a consequence, the properties of morphological neural networks are drastically different than those of traditional neural network models. In this paper we consider some of these differences and provide some particular examples of morphological neural network.

  3. Neural Tube Defects

    Science.gov (United States)

    Neural tube defects are birth defects of the brain, spine, or spinal cord. They happen in the ... that she is pregnant. The two most common neural tube defects are spina bifida and anencephaly. In ...

  4. In vitro culture and characterization of putative porcine embryonic germ cells derived from domestic breeds and yucatan mini pig embryos at days 20-24 of gestation

    DEFF Research Database (Denmark)

    Petkov, Stoyan Gueorguiev; Marks, Hendrik; Klein, Tino

    2011-01-01

    Embryonic germ cells (EGC) are cultured pluripotent cells derived from primordial germ cells (PGC). This study explored the possibility of establishing porcine EGC from domestic breeds and Yucatan mini pigs using embryos at Days 17-24 of gestation. In vitro culture of PGC from both pooled...

  5. Single-cell-derived mesenchymal stem cells overexpressing Csx/Nkx2.5 and GATA4 undergo the stochastic cardiomyogenic fate and behave like transient amplifying cells

    International Nuclear Information System (INIS)

    Yamada, Yoji; Sakurada, Kazuhiro; Takeda, Yukiji; Gojo, Satoshi; Umezawa, Akihiro

    2007-01-01

    Bone marrow-derived stromal cells can give rise to cardiomyocytes as well as adipocytes, osteocytes, and chondrocytes in vitro. The existence of mesenchymal stem cells has been proposed, but it remains unclear if a single-cell-derived stem cell stochastically commits toward a cardiac lineage. By single-cell marking, we performed a follow-up study of individual cells during the differentiation of 9-15c mesenchymal stromal cells derived from bone marrow cells. Three types of cells, i.e., cardiac myoblasts, cardiac progenitors and multipotent stem cells were differentiated from a single cell, implying that cardiomyocytes are generated stochastically from a single-cell-derived stem cell. We also demonstrated that overexpression of Csx/Nkx2.5 and GATA4, precardiac mesodermal transcription factors, enhanced cardiomyogenic differentiation of 9-15c cells, and the frequency of cardiomyogenic differentiation was increased by co-culturing with fetal cardiomyocytes. Single-cell-derived mesenchymal stem cells overexpressing Csx/Nkx2.5 and GATA4 behaved like cardiac transient amplifying cells, and still retained their plasticity in vivo

  6. Neural tissue-spheres

    DEFF Research Database (Denmark)

    Andersen, Rikke K; Johansen, Mathias; Blaabjerg, Morten

    2007-01-01

    By combining new and established protocols we have developed a procedure for isolation and propagation of neural precursor cells from the forebrain subventricular zone (SVZ) of newborn rats. Small tissue blocks of the SVZ were dissected and propagated en bloc as free-floating neural tissue...... content, thus allowing experimental studies of neural precursor cells and their niche...

  7. Cracking the Neural Code for Sensory Perception by Combining Statistics, Intervention, and Behavior.

    Science.gov (United States)

    Panzeri, Stefano; Harvey, Christopher D; Piasini, Eugenio; Latham, Peter E; Fellin, Tommaso

    2017-02-08

    The two basic processes underlying perceptual decisions-how neural responses encode stimuli, and how they inform behavioral choices-have mainly been studied separately. Thus, although many spatiotemporal features of neural population activity, or "neural codes," have been shown to carry sensory information, it is often unknown whether the brain uses these features for perception. To address this issue, we propose a new framework centered on redefining the neural code as the neural features that carry sensory information used by the animal to drive appropriate behavior; that is, the features that have an intersection between sensory and choice information. We show how this framework leads to a new statistical analysis of neural activity recorded during behavior that can identify such neural codes, and we discuss how to combine intersection-based analysis of neural recordings with intervention on neural activity to determine definitively whether specific neural activity features are involved in a task. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Neural electrical activity and neural network growth.

    Science.gov (United States)

    Gafarov, F M

    2018-05-01

    The development of central and peripheral neural system depends in part on the emergence of the correct functional connectivity in its input and output pathways. Now it is generally accepted that molecular factors guide neurons to establish a primary scaffold that undergoes activity-dependent refinement for building a fully functional circuit. However, a number of experimental results obtained recently shows that the neuronal electrical activity plays an important role in the establishing of initial interneuronal connections. Nevertheless, these processes are rather difficult to study experimentally, due to the absence of theoretical description and quantitative parameters for estimation of the neuronal activity influence on growth in neural networks. In this work we propose a general framework for a theoretical description of the activity-dependent neural network growth. The theoretical description incorporates a closed-loop growth model in which the neural activity can affect neurite outgrowth, which in turn can affect neural activity. We carried out the detailed quantitative analysis of spatiotemporal activity patterns and studied the relationship between individual cells and the network as a whole to explore the relationship between developing connectivity and activity patterns. The model, developed in this work will allow us to develop new experimental techniques for studying and quantifying the influence of the neuronal activity on growth processes in neural networks and may lead to a novel techniques for constructing large-scale neural networks by self-organization. Copyright © 2018 Elsevier Ltd. All rights reserved.

  9. Visceral fat area is a strong predictor of leukocyte cell-derived chemotaxin 2, a potential biomarker of dyslipidemia.

    Directory of Open Access Journals (Sweden)

    Kumpei Tanisawa

    Full Text Available Leukocyte cell-derived chemotaxin 2 (LECT2 is a hepatokine linking obesity to skeletal muscle insulin resistance. Although previous studies reported that obesity was associated with high levels of circulating LECT2 in human, the associations of detailed body fat distribution with LECT2 levels have not been examined. Furthermore, although animal study suggested that exercise decreased circulating LECT2 levels, it remains unknown whether physical fitness is associated with LECT2 levels in human. We therefore examined the relationship of plasma LECT2 levels with various adiposity indices and cardiorespiratory fitness (CRF in middle-aged and elderly Japanese men. Furthermore, we examined the relationship of LECT2 levels with the presence of metabolic syndrome, hypertension, insulin resistance and dyslipidemia to determine the clinical significance of measuring circulating LECT2.This was a cross-sectional study of 143 Japanese men (age: 30-79 years. Participants' plasma LECT2 levels were measured by an enzyme-linked immunosorbent assay. To assess their abdominal fat distributions, visceral fat area (VFA and subcutaneous fat area (SFA were measured using magnetic resonance imaging. CRF was assessed by measuring peak oxygen uptake ([Formula: see text].All adiposity indices measured in this study were positively correlated with plasma LECT2 levels, while [Formula: see text] was negatively correlated with LECT2 levels after adjustment for age. The correlations, except for VFA were no longer significant with further adjustment for VFA. Stepwise multiple linear regression analysis revealed that VFA was the strongest predictor of plasma LECT2 levels. Plasma LECT2 levels differed based on the presence of metabolic syndrome and dyslipidemia, but not hypertension and insulin resistance. Logistic regression analyses revealed that plasma LECT2 levels were significantly associated with dyslipidemia independently of VFA; VFA was not significantly associated with

  10. Transplantation of dedifferentiated fat cell-derived micromass pellets contributed to cartilage repair in the rat osteochondral defect model.

    Science.gov (United States)

    Shimizu, Manabu; Matsumoto, Taro; Kikuta, Shinsuke; Ohtaki, Munenori; Kano, Koichiro; Taniguchi, Hiroaki; Saito, Shu; Nagaoka, Masahiro; Tokuhashi, Yasuaki

    2018-03-20

    Mature adipocyte-derived dedifferentiated fat (DFAT) cells possesses the ability to proliferate effectively and the potential to differentiate into multiple linages of mesenchymal tissue; similar to adipose-derived stem cells (ASCs). The purpose of this study is to examine the effects of DFAT cell transplantation on cartilage repair in a rat model of osteochondral defects. Full-thickness osteochondral defects were created in the knees of Sprague-Dawley rats bilaterally. Cartilage-like micromass pellets were prepared from green fluorescent protein (GFP)-labeled rat DFAT cells and subsequently transplanted into the affected right knee of these rats. Defects in the left knee were used as a control. Macroscopic and microscopic changes of treated and control defects were evaluated up to 12 weeks post-treatment with DFAT cells. To observe the transplanted cells, sectioned femurs were immunostained for GFP and type II collagen. DFAT cells formed micromass pellets expressing characteristics of immature cartilage in vitro. In the DFAT cell-transplanted limbs, the defects were completely filled with white micromass pellets as early as 2 weeks post-treatment. These limbs became smooth at 4 weeks. Conversely, the defects in the control limbs were still not repaired by 4 weeks. Macroscopic ICRS scores at 2 and 4 weeks were significantly higher in the DFAT cells-transplanted limbs compared to those of the control limbs. The modified O'Driscol histological scores for the DFAT cell-transplanted limbs were significantly higher than those of the control limbs at corresponding time points. GFP-positive DAFT cells were detected in the transplanted area at 2 weeks but hardly visible at 12 weeks post-operation. Transplantation of DFAT cell-derived micromass pellets contribute to cartilage repair in a rat osteochondral defect model. DFAT cell transplantation may be a viable therapeutic strategy for the repair of osteochondral injuries. Copyright © 2018 The Authors. Published by

  11. Availability of human induced pluripotent stem cell-derived cardiomyocytes in assessment of drug potential for QT prolongation

    International Nuclear Information System (INIS)

    Nozaki, Yumiko; Honda, Yayoi; Tsujimoto, Shinji; Watanabe, Hitoshi; Kunimatsu, Takeshi; Funabashi, Hitoshi

    2014-01-01

    Field potential duration (FPD) in human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs), which can express QT interval in an electrocardiogram, is reported to be a useful tool to predict K + channel and Ca 2+ channel blocker effects on QT interval. However, there is no report showing that this technique can be used to predict multichannel blocker potential for QT prolongation. The aim of this study is to show that FPD from MEA (Multielectrode array) of hiPS-CMs can detect QT prolongation induced by multichannel blockers. hiPS-CMs were seeded onto MEA and FPD was measured for 2 min every 10 min for 30 min after drug exposure for the vehicle and each drug concentration. I Kr and I Ks blockers concentration-dependently prolonged corrected FPD (FPDc), whereas Ca 2+ channel blockers concentration-dependently shortened FPDc. Also, the multichannel blockers Amiodarone, Paroxetine, Terfenadine and Citalopram prolonged FPDc in a concentration dependent manner. Finally, the I Kr blockers, Terfenadine and Citalopram, which are reported to cause Torsade de Pointes (TdP) in clinical practice, produced early afterdepolarization (EAD). hiPS-CMs using MEA system and FPDc can predict the effects of drug candidates on QT interval. This study also shows that this assay can help detect EAD for drugs with TdP potential. - Highlights: • We focused on hiPS-CMs to replace in vitro assays in preclinical screening studies. • hiPS-CMs FPD is useful as an indicator to predict drug potential for QT prolongation. • MEA assay can help detect EAD for drugs with TdP potentials. • MEA assay in hiPS-CMs is useful for accurately predicting drug TdP risk in humans

  12. Availability of human induced pluripotent stem cell-derived cardiomyocytes in assessment of drug potential for QT prolongation

    Energy Technology Data Exchange (ETDEWEB)

    Nozaki, Yumiko, E-mail: yumiko-nozaki@ds-pharma.co.jp [Preclinical Research Laboratories, Dainippon Sumitomo Pharma. Co., Ltd., Suita, Osaka 564-0053 (Japan); Honda, Yayoi, E-mail: yayoi-honda@ds-pharma.co.jp [Preclinical Research Laboratories, Dainippon Sumitomo Pharma. Co., Ltd., Suita, Osaka 564-0053 (Japan); Tsujimoto, Shinji, E-mail: shinji-tsujimoto@ds-pharma.co.jp [Regenerative and Cellular Medicine Office, Dainippon Sumitomo Pharma. Co., Ltd., Chuo-ku, Tokyo 104-0031 (Japan); Watanabe, Hitoshi, E-mail: hitoshi-1-watanabe@ds-pharma.co.jp [Preclinical Research Laboratories, Dainippon Sumitomo Pharma. Co., Ltd., Suita, Osaka 564-0053 (Japan); Kunimatsu, Takeshi, E-mail: takeshi-kunimatsu@ds-pharma.co.jp [Preclinical Research Laboratories, Dainippon Sumitomo Pharma. Co., Ltd., Suita, Osaka 564-0053 (Japan); Funabashi, Hitoshi, E-mail: hitoshi-funabashi@ds-pharma.co.jp [Preclinical Research Laboratories, Dainippon Sumitomo Pharma. Co., Ltd., Suita, Osaka 564-0053 (Japan)

    2014-07-01

    Field potential duration (FPD) in human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs), which can express QT interval in an electrocardiogram, is reported to be a useful tool to predict K{sup +} channel and Ca{sup 2+} channel blocker effects on QT interval. However, there is no report showing that this technique can be used to predict multichannel blocker potential for QT prolongation. The aim of this study is to show that FPD from MEA (Multielectrode array) of hiPS-CMs can detect QT prolongation induced by multichannel blockers. hiPS-CMs were seeded onto MEA and FPD was measured for 2 min every 10 min for 30 min after drug exposure for the vehicle and each drug concentration. I{sub Kr} and I{sub Ks} blockers concentration-dependently prolonged corrected FPD (FPDc), whereas Ca{sup 2+} channel blockers concentration-dependently shortened FPDc. Also, the multichannel blockers Amiodarone, Paroxetine, Terfenadine and Citalopram prolonged FPDc in a concentration dependent manner. Finally, the I{sub Kr} blockers, Terfenadine and Citalopram, which are reported to cause Torsade de Pointes (TdP) in clinical practice, produced early afterdepolarization (EAD). hiPS-CMs using MEA system and FPDc can predict the effects of drug candidates on QT interval. This study also shows that this assay can help detect EAD for drugs with TdP potential. - Highlights: • We focused on hiPS-CMs to replace in vitro assays in preclinical screening studies. • hiPS-CMs FPD is useful as an indicator to predict drug potential for QT prolongation. • MEA assay can help detect EAD for drugs with TdP potentials. • MEA assay in hiPS-CMs is useful for accurately predicting drug TdP risk in humans.

  13. Generation of corneal epithelial cells from induced pluripotent stem cells derived from human dermal fibroblast and corneal limbal epithelium.

    Directory of Open Access Journals (Sweden)

    Ryuhei Hayashi

    Full Text Available Induced pluripotent stem (iPS cells can be established from somatic cells. However, there is currently no established strategy to generate corneal epithelial cells from iPS cells. In this study, we investigated whether corneal epithelial cells could be differentiated from iPS cells. We tested 2 distinct sources: human adult dermal fibroblast (HDF-derived iPS cells (253G1 and human adult corneal limbal epithelial cells (HLEC-derived iPS cells (L1B41. We first established iPS cells from HLEC by introducing the Yamanaka 4 factors. Corneal epithelial cells were successfully induced from the iPS cells by the stromal cell-derived inducing activity (SDIA differentiation method, as Pax6(+/K12(+ corneal epithelial colonies were observed after prolonged differentiation culture (12 weeks or later in both the L1B41 and 253G1 iPS cells following retinal pigment epithelial and lens cell induction. Interestingly, the corneal epithelial differentiation efficiency was higher in L1B41 than in 253G1. DNA methylation analysis revealed that a small proportion of differentially methylated regions still existed between L1B41 and 253G1 iPS cells even though no significant difference in methylation status was detected in the specific corneal epithelium-related genes such as K12, K3, and Pax6. The present study is the first to demonstrate a strategy for corneal epithelial cell differentiation from human iPS cells, and further suggests that the epigenomic status is associated with the propensity of iPS cells to differentiate into corneal epithelial cells.

  14. Adrenergic Stress Protection of Human iPS Cell-Derived Cardiomyocytes by Fast Kv7.1 Recycling

    Directory of Open Access Journals (Sweden)

    Ilaria Piccini

    2017-09-01

    Full Text Available The fight-or-flight response (FFR, a physiological acute stress reaction, involves positive chronotropic and inotropic effects on heart muscle cells mediated through β-adrenoceptor activation. Increased systolic calcium is required to enable stronger heart contractions whereas elevated potassium currents are to limit the duration of the action potentials and prevent arrhythmia. The latter effect is accomplished by an increased functional activity of the Kv7.1 channel encoded by KCNQ1. Current knowledge, however, does not sufficiently explain the full extent of rapid Kv7.1 activation and may hence be incomplete. Using inducible genetic KCNQ1 complementation in KCNQ1-deficient human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs, we here reinvestigate the functional role of Kv7.1 in adapting human CMs to adrenergic stress. Under baseline conditions, Kv7.1 was barely detectable at the plasma membrane of hiPSC-CMs, yet it fully protected these from adrenergic stress-induced beat-to-beat variability of repolarization and torsade des pointes-like arrhythmia. Furthermore, isoprenaline treatment increased field potential durations specifically in KCNQ1-deficient CMs to cause these adverse macroscopic effects. Mechanistically, we find that the protective action by Kv7.1 resides in a rapid translocation of channel proteins from intracellular stores to the plasma membrane, induced by adrenergic signaling. Gene silencing experiments targeting RAB GTPases, mediators of intracellular vesicle trafficking, showed that fast Kv7.1 recycling under acute stress conditions is RAB4A-dependent.Our data reveal a key mechanism underlying the rapid adaptation of human cardiomyocytes to adrenergic stress. These findings moreover aid to the understanding of disease pathology in long QT syndrome and bear important implications for safety pharmacological screening.

  15. Hepatic ischemia and reperfusion injury in the absence of myeloid cell-derived COX-2 in mice.

    Directory of Open Access Journals (Sweden)

    Sergio Duarte

    Full Text Available Cyclooxygenase-2 (COX-2 is a mediator of hepatic ischemia and reperfusion injury (IRI. While both global COX-2 deletion and pharmacologic COX-2 inhibition ameliorate liver IRI, the clinical use of COX-2 inhibitors has been linked to increased risks of heart attack and stroke. Therefore, a better understanding of the role of COX-2 in different cell types may lead to improved therapeutic strategies for hepatic IRI. Macrophages of myeloid origin are currently considered to be important sources of the COX-2 in damaged livers. Here, we used a Cox-2flox conditional knockout mouse (COX-2-M/-M to examine the function of COX-2 expression in myeloid cells during liver IRI. COX-2-M/-M mice and their WT control littermates were subjected to partial liver ischemia followed by reperfusion. COX-2-M/-M macrophages did not express COX-2 upon lipopolysaccharide stimulation and COX-2-M/-M livers showed reduced levels of COX-2 protein post-IRI. Nevertheless, selective deletion of myeloid cell-derived COX-2 failed to ameliorate liver IRI; serum transaminases and histology were comparable in both COX-2-M/-M and WT mice. COX-2-M/-M livers, like WT livers, developed extensive necrosis, vascular congestion, leukocyte infiltration and matrix metalloproteinase-9 (MMP-9 expression post-reperfusion. In addition, myeloid COX-2 deletion led to a transient increase in IL-6 levels after hepatic reperfusion, when compared to controls. Administration of celecoxib, a selective COX-2 inhibitor, resulted in significantly improved liver function and histology in both COX-2-M/-M and WT mice post-reperfusion, providing evidence that COX-2-mediated liver IRI is caused by COX-2 derived from a source(s other than myeloid cells. In conclusion, these results support the view that myeloid COX-2, including myeloid-macrophage COX-2, is not responsible for the hepatic IRI phenotype.

  16. Induction of Protective Immunity against Eimeria tenella, Eimeria maxima, and Eimeria acervulina Infections Using Dendritic Cell-Derived Exosomes

    Science.gov (United States)

    Gallego, Margarita; Lee, Sung Hyen; Lillehoj, Hyun Soon; Quilez, Joaquin; Lillehoj, Erik P.; Sánchez-Acedo, Caridad

    2012-01-01

    This study describes a novel immunization strategy against avian coccidiosis using exosomes derived from Eimeria parasite antigen (Ag)-loaded dendritic cells (DCs). Chicken intestinal DCs were isolated and pulsed in vitro with a mixture of sporozoite-extracted Ags from Eimeria tenella, E. maxima, and E. acervulina, and the cell-derived exosomes were isolated. Chickens were nonimmunized or immunized intramuscularly with exosomes and subsequently noninfected or coinfected with E. tenella, E. maxima, and E. acervulina oocysts. Immune parameters compared among the nonimmunized/noninfected, nonimmunized/infected, and immunized/infected groups were the numbers of cells secreting Th1 cytokines, Th2 cytokines, interleukin-16 (IL-16), and Ag-reactive antibodies in vitro and in vivo readouts of protective immunity against Eimeria infection. Cecal tonsils, Peyer's patches, and spleens of immunized and infected chickens had increased numbers of cells secreting the IL-16 and the Th1 cytokines IL-2 and gamma interferon, greater Ag-stimulated proliferative responses, and higher numbers of Ag-reactive IgG- and IgA-producing cells following in vitro stimulation with the sporozoite Ags compared with the nonimmunized/noninfected and nonimmunized/infected controls. In contrast, the numbers of cells secreting the Th2 cytokines IL-4 and IL-10 were diminished in immunized and infected chickens compared with the nonimmunized/noninfected and the nonimmunized/infected controls. Chickens immunized with Ag-loaded exosomes and infected in vivo with Eimeria oocysts had increased body weight gains, reduced feed conversion ratios, diminished fecal oocyst shedding, lessened intestinal lesion scores, and reduced mortality compared with the nonimmunized/infected controls. These results suggest that successful field vaccination against avian coccidiosis using exosomes derived from DCs incubated with Ags isolated from Eimeria species may be possible. PMID:22354026

  17. Advanced Good Cell Culture Practice for human primary, stem cell-derived and organoid models as well as microphysiological systems.

    Science.gov (United States)

    Pamies, David; Bal-Price, Anna; Chesné, Christophe; Coecke, Sandra; Dinnyes, Andras; Eskes, Chantra; Grillari, Regina; Gstraunthaler, Gerhard; Hartung, Thomas; Jennings, Paul; Leist, Marcel; Martin, Ulrich; Passier, Robert; Schwamborn, Jens C; Stacey, Glyn N; Ellinger-Ziegelbauer, Heidrun; Daneshian, Mardas

    2018-04-13

    A major reason for the current reproducibility crisis in the life sciences is the poor implementation of quality control measures and reporting standards. Improvement is needed, especially regarding increasingly complex in vitro methods. Good Cell Culture Practice (GCCP) was an effort from 1996 to 2005 to develop such minimum quality standards also applicable in academia. This paper summarizes recent key developments in in vitro cell culture and addresses the issues resulting for GCCP, e.g. the development of induced pluripotent stem cells (iPSCs) and gene-edited cells. It further deals with human stem-cell-derived models and bioengineering of organo-typic cell cultures, including organoids, organ-on-chip and human-on-chip approaches. Commercial vendors and cell banks have made human primary cells more widely available over the last decade, increasing their use, but also requiring specific guidance as to GCCP. The characterization of cell culture systems including high-content imaging and high-throughput measurement technologies increasingly combined with more complex cell and tissue cultures represent a further challenge for GCCP. The increasing use of gene editing techniques to generate and modify in vitro culture models also requires discussion of its impact on GCCP. International (often varying) legislations and market forces originating from the commercialization of cell and tissue products and technologies are further impacting on the need for the use of GCCP. This report summarizes the recommendations of the second of two workshops, held in Germany in December 2015, aiming map the challenge and organize the process or developing a revised GCCP 2.0.

  18. Resveratrol-Induced Apoptosis and Increased Radiosensitivity in CD133-Positive Cells Derived From Atypical Teratoid/Rhabdoid Tumor

    International Nuclear Information System (INIS)

    Kao, C.-L.; Huang, P.-I; Tsai, P.-H.; Tsai, M.-L.; Lo, J.-F.; Lee, Y.-Y.; Chen, Y.-J.; Chen, Y.-W.; Chiou, S.-H.

    2009-01-01

    Purpose: CD133 has recently been proposed as a marker for cancer stem-like cells (CSC) in brain tumors. The aim of the present study was to investigate the possible role of resveratrol (RV) in radiosensitivity of CD133-positive/-negative cells derived from atypical teratoid/rhabdoid tumors (AT/RT-CD133 +/- ). Materials and Methods: AT/RT-CD133 +/- were isolated and characterized by flow cytometry and quantitative real-time reverse transcription-polymerase chain reaction, and then treated with RV at different doses. Migratory ability, colony formation, apoptotic activity, and xenotransplantation were assessed for RV alone, ionizing radiation (IR) alone, and IR with RV conditions. Results: AT/RT-CD133 + displayed enhanced self-renewal and highly coexpressed 'stem cell' genes and drug-resistant genes, in addition to showing significant resistance to chemotherapeutic agents and radiotherapy as compared with CD133 - cells. After treatment with 200 μM RV, the in vitro proliferation rates and in vivo tumor restoration abilities of ATRT-CD133 + were dramatically inhibited. Importantly, treatment with 150 μM RV can effectively inhibit the expression of drug-resistant genes in AT/RT-CD133 + , and further facilitate to the differentiation of CD133 + into CD133 - . In addition, treatment with 150 μM RV could significantly enhance the radiosensitivity and IR-mediated apoptosis in RV-treated ATRT-CD133 +/- . Kaplan-Meier survival analysis indicated that the mean survival rate of mice with ATRT-CD133 + that were treated with IR could be significantly improved when IR was combined with 150 μM RV treatment. Conclusions: AT/RT-CD133 + exhibit CSC properties and are refractory to IR treatment. Our results suggest that RV treatment plays crucial roles in antiproliferative, proapoptotic, and radiosensitizing effects on treated-CD133 +/- ; RV may therefore improve the clinical treatment of AT/RT.

  19. An Abbreviated Protocol for In Vitro Generation of Functional Human Embryonic Stem Cell-Derived Beta-Like Cells.

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    Mohammad Massumi

    Full Text Available The ability to yield glucose-responsive pancreatic beta-cells from human pluripotent stem cells in vitro will facilitate the development of the cell replacement therapies for the treatment of Type 1 Diabetes. Here, through the sequential in vitro targeting of selected signaling pathways, we have developed an abbreviated five-stage protocol (25-30 days to generate human Embryonic Stem Cell-Derived Beta-like Cells (ES-DBCs. We showed that Geltrex, as an extracellular matrix, could support the generation of ES-DBCs more efficiently than that of the previously described culture systems. The activation of FGF and Retinoic Acid along with the inhibition of BMP, SHH and TGF-beta led to the generation of 75% NKX6.1+/NGN3+ Endocrine Progenitors. The inhibition of Notch and tyrosine kinase receptor AXL, and the treatment with Exendin-4 and T3 in the final stage resulted in 35% mono-hormonal insulin positive cells, 1% insulin and glucagon positive cells and 30% insulin and NKX6.1 co-expressing cells. Functionally, ES-DBCs were responsive to high glucose in static incubation and perifusion studies, and could secrete insulin in response to successive glucose stimulations. Mitochondrial metabolic flux analyses using Seahorse demonstrated that the ES-DBCs could efficiently metabolize glucose and generate intracellular signals to trigger insulin secretion. In conclusion, targeting selected signaling pathways for 25-30 days was sufficient to generate ES-DBCs in vitro. The ability of ES-DBCs to secrete insulin in response to glucose renders them a promising model for the in vitro screening of drugs, small molecules or genes that may have potential to influence beta-cell function.

  20. Cytokeratin expression of engrafted three-dimensional culture tissues using epithelial cells derived from porcine periodontal ligaments.

    Science.gov (United States)

    Yamada, Rie; Kitajima, Kayoko; Arai, Kyoko; Igarashi, Masaru

    2014-09-01

    This study investigated the differentiation and proliferation of epithelial cells derived from periodontal ligaments after three-dimensional culture using collagen gel with fibroblasts in vitro and in vivo. Epithelial cells and fibroblasts were derived from porcine periodontal ligaments. Epithelial cells were labeled using a fluorescent red membrane marker (PKH-26GL) and were seeded onto collagen gel with fibroblasts, followed by incubation in an air-liquid interface for 7 days. Three-dimensional cultures were grafted onto the backs of nude mice and removed at 1, 7, and 14 days after surgery (in vivo model). Unfixed sections (5 μm) were used to detect the presence of red fluorescent cells. Paraffin sections were analyzed histologically and immunohistochemically. Specimens were compared with three-dimensional culture tissues at 8, 14 and 21 days (in vitro model). Grafted three-dimensional cultures formed a stratified epithelial structure similar to skin in vivo. Epithelial cells were sequenced in basal-layer-like structures at 14 days in vivo. Immunohistochemical findings showed that the expression of cytokeratin was detected in the epithelial layer in in vitro and in vivo models. Ck8 + 18 + 19 was expressed in the upper epithelial layer in the in vitro model at 14 and 21 days, but not in vivo. Involucrin was expressed in the certified layers in vitro at 14 days, but not in vivo. Laminin was detected at the dermo-epidermal junction in vivo at 7 and 14 days, but not in vitro. These results suggest that differentiation of three-dimensional culture tissues differs in vivo and in vitro. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  1. Evaluation of the cardiotoxicity of mitragynine and its analogues using human induced pluripotent stem cell-derived cardiomyocytes.

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    Jun Lu

    Full Text Available Mitragynine is a major bioactive compound of Kratom, which is derived from the leave extracts of Mitragyna speciosa Korth or Mitragyna speciosa (M. speciosa, a medicinal plant from South East Asia used legally in many countries as stimulant with opioid-like effects for the treatment of chronic pain and opioid-withdrawal symptoms. Fatal incidents with Mitragynine have been associated with cardiac arrest. In this study, we determined the cardiotoxicity of Mitragynine and other chemical constituents isolated using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs.The rapid delayed rectifier potassium current (IKr, L-type Ca2+ current (ICa,L and action potential duration (APD were measured by whole cell patch-clamp. The expression of KCNH2 and cytotoxicity was determined by real-time PCR and Caspase activity measurements. After significant IKr suppression by Mitragynine (10 µM was confirmed in hERG-HEK cells, we systematically examined the effects of Mitragynine and other chemical constituents in hiPSC-CMs. Mitragynine, Paynantheine, Speciogynine and Speciociliatine, dosage-dependently (0.1∼100 µM suppressed IKr in hiPSC-CMs by 67%∼84% with IC50 ranged from 0.91 to 2.47 µM. Moreover, Mitragynine (10 µM significantly prolonged APD at 50 and 90% repolarization (APD50 and APD90 (439.0±11.6 vs. 585.2±45.5 ms and 536.0±22.6 vs. 705.9±46.1 ms, respectively and induced arrhythmia, without altering the L-type Ca2+ current. Neither the expression, and intracellular distribution of KCNH2/Kv11.1, nor the Caspase 3 activity were significantly affected by Mitragynine.Our study indicates that Mitragynine and its analogues may potentiate Torsade de Pointes through inhibition of IKr in human cardiomyocytes.

  2. Linking incomplete reprogramming to the improved pluripotency of murine embryonal carcinoma cell-derived pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Gang Chang

    Full Text Available Somatic cell nuclear transfer (SCNT has been proved capable of reprogramming various differentiated somatic cells into pluripotent stem cells. Recently, induced pluripotent stem cells (iPS have been successfully derived from mouse and human somatic cells by the over-expression of a combination of transcription factors. However, the molecular mechanisms underlying the reprogramming mediated by either the SCNT or iPS approach are poorly understood. Increasing evidence indicates that many tumor pathways play roles in the derivation of iPS cells. Embryonal carcinoma (EC cells have the characteristics of both stem cells and cancer cells and thus they might be the better candidates for elucidating the details of the reprogramming process. Although previous studies indicate that EC cells cannot be reprogrammed into real pluripotent stem cells, the reasons for this remain unclear. Here, nuclei from mouse EC cells (P19 were transplanted into enucleated oocytes and pluripotent stem cells (P19 NTES cells were subsequently established. Interestingly, P19 NTES cells prolonged the development of tetraploid aggregated embryos compared to EC cells alone. More importantly, we found that the expression recovery of the imprinted H19 gene was dependent on the methylation state in the differential methylation region (DMR. The induction of Nanog expression, however, was independent of the promoter region DNA methylation state in P19 NTES cells. A whole-genome transcriptome analysis further demonstrated that P19 NTES cells were indeed the intermediates between P19 cells and ES cells and many interesting genes were uncovered that may be responsible for the failed reprogramming of P19 cells. To our knowledge, for the first time, we linked incomplete reprogramming to the improved pluripotency of EC cell-derived pluripotent stem cells. The candidate genes we discovered may be useful not only for understanding the mechanisms of reprogramming, but also for deciphering the

  3. Eighteen-Year Cryopreservation Does Not Negatively Affect the Pluripotency of Human Embryos: Evidence from Embryonic Stem Cell Derivation

    Science.gov (United States)

    Rungsiwiwut, Ruttachuk; Numchaisrika, Pranee; Ahnonkitpanit, Vichuda; Isarasena, Nipan; Virutamasen, Pramuan

    2012-01-01

    Abstract Human embryonic stem (hES) cells are considered to be a potential source for the therapy of human diseases, drug screening, and the study of developmental biology. In the present study, we successfully derived hES cell lines from blastocysts developed from frozen and fresh embryos. Seventeen- to eighteen-year-old frozen embryos were thawed, cultured to the blastocyst stage, and induced to form hES cells using human foreskin fibroblasts. The Chula2.hES cell line and the Chula4.hES and Chula5.hES cell lines were derived from blastocysts developed from frozen and fresh embryos, respectively. The cell lines expressed pluripotent markers, including alkaline phosphatase (AP), Oct3/4, stage-specific embryonic antigen (SSEA)-4, and tumor recognition antigen (TRA)-1-60 and TRA-1-81 as detected with immunocytochemistry. The real-time polymerase chain reaction (RT-PCR) results showed that the cell lines expressed pluripotent genes, including OCT3/4, SOX2, NANOG, UTF, LIN28, REX1, NODAL, and E-Cadherin. In addition, the telomerase activities of the cell lines were higher than in the fibroblast cells. Moreover, the cell lines differentiated into all three germ layers both in vitro and in vivo. The cell lines had distinct identities, as revealed with DNA fingerprinting, and maintained their normal karyotype after a long-term culture. This study is the first to report the successful derivation of hES cell lines in Thailand and that frozen embryos maintained their pluripotency similar to fresh embryos, as shown by the success of hES cell derivation, even after years of cryopreservation. Therefore, embryos from prolonged cryopreservation could be an alternative source for embryonic stem cell research. PMID:23514952

  4. Comparative study of regenerative effects of mesenchymal stem cells derived from placental amnion, chorion and umbilical cord on dermal wounds.

    Science.gov (United States)

    Ertl, Juliane; Pichlsberger, Melanie; Tuca, Alexandru-Cristian; Wurzer, Paul; Fuchs, Jakob; Geyer, Stefan H; Maurer-Gesek, Barbara; Weninger, Wolfgang J; Pfeiffer, Dagmar; Bubalo, Vladimir; Parvizi, Daryousch; Kamolz, Lars-Peter; Lang, Ingrid

    2018-05-01

    Mesenchymal stem/stromal cells derived from human term placentas (PMSCs) are novel therapeutic agents and more topical than ever. Here we evaluated the effects of three types of PMSCs on wound healing in an in vivo mouse model: Amnion-derived MSCs (AMSCs), blood vessel-derived MSCs (BV-MSCs) from the chorionic plate and Wharton's jelly-derived MSCs (WJ-MSCs) from the umbilical cord. We topically applied PMSCs onto skin wounds in mice using the dermal substitute Matriderm ® as carrier and evaluated wound healing parameters. In addition, we investigated the effects of all PMSC types under co-application with placental endothelial cells (PLECs). After 8 days, we compared the percent of wound closure and the angiogenic potential between all groups. AMSCs, BV-MSCs and WJ-MSCs significantly induced a faster healing and a higher number of blood vessels in the wound when compared to controls (Matriderm ® -alone). PLECs did not further improve the advantageous effects of PMSC-treatment. Quantitative data and 3D analysis by high resolution episcopic microscopy confirmed a lower density of vessels in Matriderm ® /PMSCs/PLECs co-application compared to Matriderm ® /PMSCs treatment. Results indicate that all three PMSC types exert similar beneficial effects on wound closure and neovascularization in our mouse model. Using Matriderm ® as carrier for PMSCs propagates rapid cell migration towards the wound area that allows a fast and clinically practicable method for stem cell application. These promising effects warrant further investigation in clinical trials. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

  5. Connective tissue activation. XXXII. Structural and biologic characteristics of mesenchymal cell-derived connective tissue activating peptide-V.

    Science.gov (United States)

    Cabral, A R; Cole, L A; Walz, D A; Castor, C W

    1987-12-01

    Connective tissue activating peptide-V (CTAP-V) is a single-chain, mesenchymal cell-derived anionic protein with large and small molecular forms (Mr of 28,000 and 16,000, respectively), as defined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins have similar specific activities with respect to stimulation of hyaluronic acid and DNA formation in human synovial fibroblast cultures. S-carboxymethylation or removal of sialic acid residues did not modify CTAP-V biologic activity. Rabbit antibodies raised separately against each of the purified CTAP-V proteins reacted, on immunodiffusion and on Western blot, with each antigen and neutralized mitogenic activity. The amino-terminal amino acid sequence of the CTAP-V proteins, determined by 2 laboratories, confirmed their structural similarities. The amino-terminal sequence through 37 residues was demonstrated for the smaller protein. The first 10 residues of CTAP-V (28 kd) were identical to the N-terminal decapeptide of CTAP-V (16 kd). The C-terminal sequence, determined by carboxypeptidase Y digestion, was the same for both CTAP-V molecular species. The 2 CTAP-V peptides had similar amino acid compositions, whether residues were expressed as a percent of the total or were normalized to mannose. Reduction of native CTAP-V protein released sulfhydryl groups in a protein:disulfide ratio of 1:2; this suggests that CTAP-V contains 2 intramolecular disulfide bonds. Clearly, CTAP-V is a glycoprotein. The carbohydrate content of CTAP-V (16 kd) and CTAP-V (28 kd) is 27% and 25%, respectively. CTAP-V may have significance in relation to autocrine mechanisms for growth regulation of connective tissue cells and other cell types.

  6. The absolute counting of red cell-derived microparticles with red cell bead by flow rate based assay.

    Science.gov (United States)

    Nantakomol, Duangdao; Imwong, Malika; Soontarawirat, Ingfar; Kotjanya, Duangporn; Khakhai, Chulalak; Ohashi, Jun; Nuchnoi, Pornlada

    2009-05-01

    Activation of red blood cell is associated with the formation of red cell-derived microparticles (RMPs). Analysis of circulating RMPs is becoming more refined and clinically useful. A quantitative Trucount tube method is the conventional method uses for quantitating RMPs. In this study, we validated a quantitative method called "flow rate based assay using red cell bead (FCB)" to measure circulating RMPs in the peripheral blood of healthy subjects. Citrated blood samples collected from 30 cases of healthy subjects were determined the RMPs count by using double labeling of annexin V-FITC and anti-glycophorin A-PE. The absolute RMPs numbers were measured by FCB, and the results were compared with the Trucount or with flow rate based calibration (FR). Statistical correlation and agreement were analyzed using linear regression and Bland-Altman analysis. There was no significant difference in the absolute number of RMPs quantitated by FCB when compared with those two reference methods including the Trucount tube and FR method. The absolute RMPs count obtained from FCB method was highly correlated with those obtained from Trucount tube (r(2) = 0.98, mean bias 4 cell/microl, limit of agreement [LOA] -20.3 to 28.3 cell/microl), and FR method (r(2) = 1, mean bias 10.3 cell/microl, and LOA -5.5 to 26.2 cell/microl). This study demonstrates that FCB is suitable and more affordable for RMPs quantitation in the clinical samples. This method is a low cost and interchangeable to latex bead-based method for generating the absolute counts in the resource-limited areas. (c) 2008 Clinical Cytometry Society.

  7. Divergent effects of adrenaline in human induced pluripotent stem cell-derived cardiomyocytes obtained from hypertrophic cardiomyopathy

    Directory of Open Access Journals (Sweden)

    Chandra Prajapati

    2018-02-01

    Full Text Available Hypertrophic cardiomyopathy (HCM is a common inherited cardiac disease that affects the heart muscle with diverse clinical outcomes. HCM can cause sudden cardiac death (SCD during or immediately after mild to rigorous physical activity in young patients. However, the mechanism causing SCD as a result of exercise remains unknown, but exercise-induced ventricular arrhythmias are thought to be responsible for this fatal consequence. To understand the disease mechanism behind HCM in a better way, we generated patient-specific induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs from HCM patients carrying either the MYBPC3-Gln1061X or TPM1-Asp175Asn mutation. We extensively investigated the effects of low to high concentrations of adrenaline on action potential characteristics, and the occurrence of arrhythmias in the presence of various concentrations of adrenaline and in wash-out condition. We classified and quantified different types of arrhythmias observed in hiPSC-CMs, and found that the occurrence of arrhythmias was dependent on concentrations of adrenaline and positions of mutations in genes causing HCM. In addition, we observed ventricular tachycardia types of arrhythmias in hiPSC-CMs carrying the TPM1-Asp175Asn mutation. We additionally examined the antiarrhythmic potency of bisoprolol in HCM-specific hiPSC-CMs. However, bisoprolol could not reduce the occurrence of arrhythmias during administration or during the wash-out condition of adrenaline in HCM-specific hiPSC-CMs. Our study demonstrates hiPSC-CMs as a promising tool for studying HCM. The experimental design used in this study could be suitable and beneficial for studying other components and drugs related to cardiac disease in general.

  8. Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy

    Science.gov (United States)

    Askari, Arman T.; Unzek, Samuel; Popovic, Zoran B.; Goldman, Corey K.; Forudi, Farhad; Kiedrowski, Matthew; Rovner, Aleksandr; Ellis, Stephen G.; Thomas, James D.; DiCorleto, Paul E.; hide

    2003-01-01

    BACKGROUND: Myocardial regeneration via stem-cell mobilisation at the time of myocardial infarction is known to occur, although the mechanism for stem-cell homing to infarcted tissue subsequently and whether this approach can be used for treatment of ischaemic cardiomyopathy are unknown. We investigated these issues in a Lewis rat model (ligation of the left anterior descending artery) of ischaemic cardiomyopathy. METHODS: We studied the effects of stem-cell mobilisation by use of granulocyte colony-stimulating factor (filgrastim) with or without transplantation of syngeneic cells. Shortening fraction and myocardial strain by tissue doppler imaging were quantified by echocardiography. FINDINGS: Stem-cell mobilisation with filgrastim alone did not lead to engraftment of bone-marrow-derived cells. Stromal-cell-derived factor 1 (SDF-1), required for stem-cell homing to bone marrow, was upregulated immediately after myocardial infarction and downregulated within 7 days. 8 weeks after myocardial infarction, transplantation into the peri-infarct zone of syngeneic cardiac fibroblasts stably transfected to express SDF-1 induced homing of CD117-positive stem cells to injured myocardium after filgrastim administration (control vs SDF-1-expressing cardiac fibroblasts mean 7.2 [SD 3.4] vs 33.2 [6.0] cells/mm2, n=4 per group, pcell homing to injured myocardium and suggest a strategy for directed stem-cell engraftment into injured tissues. Our findings also indicate that therapeutic strategies focused on stem-cell mobilisation for regeneration of myocardial tissue must be initiated within days of myocardial infarction unless signalling for stem-cell homing is re-established.

  9. Visceral fat area is a strong predictor of leukocyte cell-derived chemotaxin 2, a potential biomarker of dyslipidemia.

    Science.gov (United States)

    Tanisawa, Kumpei; Taniguchi, Hirokazu; Sun, Xiaomin; Ito, Tomoko; Kawakami, Ryoko; Sakamoto, Shizuo; Higuchi, Mitsuru

    2017-01-01

    Leukocyte cell-derived chemotaxin 2 (LECT2) is a hepatokine linking obesity to skeletal muscle insulin resistance. Although previous studies reported that obesity was associated with high levels of circulating LECT2 in human, the associations of detailed body fat distribution with LECT2 levels have not been examined. Furthermore, although animal study suggested that exercise decreased circulating LECT2 levels, it remains unknown whether physical fitness is associated with LECT2 levels in human. We therefore examined the relationship of plasma LECT2 levels with various adiposity indices and cardiorespiratory fitness (CRF) in middle-aged and elderly Japanese men. Furthermore, we examined the relationship of LECT2 levels with the presence of metabolic syndrome, hypertension, insulin resistance and dyslipidemia to determine the clinical significance of measuring circulating LECT2. This was a cross-sectional study of 143 Japanese men (age: 30-79 years). Participants' plasma LECT2 levels were measured by an enzyme-linked immunosorbent assay. To assess their abdominal fat distributions, visceral fat area (VFA) and subcutaneous fat area (SFA) were measured using magnetic resonance imaging. CRF was assessed by measuring peak oxygen uptake ([Formula: see text]). All adiposity indices measured in this study were positively correlated with plasma LECT2 levels, while [Formula: see text] was negatively correlated with LECT2 levels after adjustment for age. The correlations, except for VFA were no longer significant with further adjustment for VFA. Stepwise multiple linear regression analysis revealed that VFA was the strongest predictor of plasma LECT2 levels. Plasma LECT2 levels differed based on the presence of metabolic syndrome and dyslipidemia, but not hypertension and insulin resistance. Logistic regression analyses revealed that plasma LECT2 levels were significantly associated with dyslipidemia independently of VFA; VFA was not significantly associated with dyslipidemia

  10. Role of mast cell- and non-mast cell-derived inflammatory mediators in immunologic induction of synaptic plasticity

    Directory of Open Access Journals (Sweden)

    A.A.C. Albuquerque

    1997-07-01

    Full Text Available We have previously discovered a long-lasting enhancement of synaptic transmission in mammal autonomic ganglia caused by immunological activation of ganglionic mast cells. Subsequent to mast cell activation, lipid and peptide mediators are released which may modulate synaptic function. In this study we determined whether some mast cell-derived mediators, prostaglandin D2 (PGD2; 1.0 µM, platelet aggregating factor (PAF; 0.3 µM and U44619 (a thromboxane analogue; 1.0 µM, and also endothelin-1 (ET-1; 0.5 µM induce synaptic potentiation in the guinea pig superior cervical ganglion (SCG, and compared their effects on synaptic transmission with those induced by a sensitizing antigen, ovalbumin (OVA; 10 µg/ml. The experiments were carried out on SCGs isolated from adult male guinea pigs (200-250 g actively sensitized to OVA, maintained in oxygenated Locke solution at 37oC. Synaptic potentiation was measured through alterations of the integral of the post-ganglionic compound action potential (CAP. All agents tested caused long-term (LTP; duration ³30 min or short-term (STP; <30 min potentiation of synaptic efficacy, as measured by the increase in the integral of the post-ganglionic CAP. The magnitude of mediator-induced potentiation was never the same as the antigen-induced long-term potentiation (A-LTP. The agent that best mimicked the antigen was PGD2, which induced a 75% increase in CAP integral for LTP (antigen: 94% and a 34% increase for STP (antigen: 91%. PAF-, U44619-, and ET-1-induced increases in CAP integral ranged for LTP from 34 to 47%, and for STP from 0 to 26%. These results suggest that the agents investigated may participate in the induction of A-LTP

  11. Patch-Clamp Recording from Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Improving Action Potential Characteristics through Dynamic Clamp

    Science.gov (United States)

    Veerman, Christiaan C.; Zegers, Jan G.; Mengarelli, Isabella; Bezzina, Connie R.

    2017-01-01

    Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold great promise for studying inherited cardiac arrhythmias and developing drug therapies to treat such arrhythmias. Unfortunately, until now, action potential (AP) measurements in hiPSC-CMs have been hampered by the virtual absence of the inward rectifier potassium current (IK1) in hiPSC-CMs, resulting in spontaneous activity and altered function of various depolarising and repolarising membrane currents. We assessed whether AP measurements in “ventricular-like” and “atrial-like” hiPSC-CMs could be improved through a simple, highly reproducible dynamic clamp approach to provide these cells with a substantial IK1 (computed in real time according to the actual membrane potential and injected through the patch-clamp pipette). APs were measured at 1 Hz using perforated patch-clamp methodology, both in control cells and in cells treated with all-trans retinoic acid (RA) during the differentiation process to increase the number of cells with atrial-like APs. RA-treated hiPSC-CMs displayed shorter APs than control hiPSC-CMs and this phenotype became more prominent upon addition of synthetic IK1 through dynamic clamp. Furthermore, the variability of several AP parameters decreased upon IK1 injection. Computer simulations with models of ventricular-like and atrial-like hiPSC-CMs demonstrated the importance of selecting an appropriate synthetic IK1. In conclusion, the dynamic clamp-based approach of IK1 injection has broad applicability for detailed AP measurements in hiPSC-CMs. PMID:28867785

  12. Generation and properties of a new human ventral mesencephalic neural stem cell line

    DEFF Research Database (Denmark)

    Villa, Ana; Liste, Isabel; Courtois, Elise T

    2009-01-01

    . Here we report the generation of a new stable cell line of human neural stem cells derived from ventral mesencephalon (hVM1) based on v-myc immortalization. The cells expressed neural stem cell and radial glia markers like nestin, vimentin and 3CB2 under proliferation conditions. After withdrawal......Neural stem cells (NSCs) are powerful research tools for the design and discovery of new approaches to cell therapy in neurodegenerative diseases like Parkinson's disease. Several epigenetic and genetic strategies have been tested for long-term maintenance and expansion of these cells in vitro...... derivatives may constitute good candidates for the study of development and physiology of human dopaminergic neurons in vitro, and to develop tools for Parkinson's disease cell replacement preclinical research and drug testing....

  13. Chaotic diagonal recurrent neural network

    International Nuclear Information System (INIS)

    Wang Xing-Yuan; Zhang Yi

    2012-01-01

    We propose a novel neural network based on a diagonal recurrent neural network and chaos, and its structure and learning algorithm are designed. The multilayer feedforward neural network, diagonal recurrent neural network, and chaotic diagonal recurrent neural network are used to approach the cubic symmetry map. The simulation results show that the approximation capability of the chaotic diagonal recurrent neural network is better than the other two neural networks. (interdisciplinary physics and related areas of science and technology)

  14. Evolvable Neural Software System

    Science.gov (United States)

    Curtis, Steven A.

    2009-01-01

    The Evolvable Neural Software System (ENSS) is composed of sets of Neural Basis Functions (NBFs), which can be totally autonomously created and removed according to the changing needs and requirements of the software system. The resulting structure is both hierarchical and self-similar in that a given set of NBFs may have a ruler NBF, which in turn communicates with other sets of NBFs. These sets of NBFs may function as nodes to a ruler node, which are also NBF constructs. In this manner, the synthetic neural system can exhibit the complexity, three-dimensional connectivity, and adaptability of biological neural systems. An added advantage of ENSS over a natural neural system is its ability to modify its core genetic code in response to environmental changes as reflected in needs and requirements. The neural system is fully adaptive and evolvable and is trainable before release. It continues to rewire itself while on the job. The NBF is a unique, bilevel intelligence neural system composed of a higher-level heuristic neural system (HNS) and a lower-level, autonomic neural system (ANS). Taken together, the HNS and the ANS give each NBF the complete capabilities of a biological neural system to match sensory inputs to actions. Another feature of the NBF is the Evolvable Neural Interface (ENI), which links the HNS and ANS. The ENI solves the interface problem between these two systems by actively adapting and evolving from a primitive initial state (a Neural Thread) to a complicated, operational ENI and successfully adapting to a training sequence of sensory input. This simulates the adaptation of a biological neural system in a developmental phase. Within the greater multi-NBF and multi-node ENSS, self-similar ENI s provide the basis for inter-NBF and inter-node connectivity.

  15. Induced Pluripotent Stem Cells Derived from a CLN5 Patient Manifest Phenotypic Characteristics of Neuronal Ceroid Lipofuscinoses

    Directory of Open Access Journals (Sweden)

    Kristiina Uusi-Rauva

    2017-05-01

    Full Text Available Neuronal ceroid lipofuscinoses (NCLs are autosomal recessive progressive encephalopathies caused by mutations in at least 14 different genes. Despite extensive studies performed in different NCL animal models, the molecular mechanisms underlying neurodegeneration in NCLs remain poorly understood. To model NCL in human cells, we generated induced pluripotent stem cells (iPSCs by reprogramming skin fibroblasts from a patient with CLN5 (ceroid lipofuscinosis, neuronal, 5 disease, the late infantile variant form of NCL. These CLN5 patient-derived iPSCs (CLN5Y392X iPSCs harbouring the most common CLN5 mutation, c.1175_1176delAT (p.Tyr392X, were further differentiated into neural lineage cells, the most affected cell type in NCLs. The CLN5Y392X iPSC-derived neural lineage cells showed accumulation of autofluorescent storage material and subunit C of the mitochondrial ATP synthase, both representing the hallmarks of many forms of NCLs, including CLN5 disease. In addition, we detected abnormalities in the intracellular organelles and aberrations in neuronal sphingolipid transportation, verifying the previous findings obtained from Cln5-deficient mouse macrophages. Therefore, patient-derived iPSCs provide a suitable model to study the mechanisms of NCL diseases.

  16. Chemokines, macrophage inflammatory protein-2 and stromal cell-derived factor-1{alpha}, suppress amyloid {beta}-induced neurotoxicity

    Energy Technology Data Exchange (ETDEWEB)

    Raman, Dayanidhi; Milatovic, Snjezana-Zaja [Department of Cancer Biology, Vanderbilt University, School of Medicine, Nashville, TN 37232 (United States); Milatovic, Dejan [Department of Pediatrics/Pediatric Toxicology, Vanderbilt University, School of Medicine, Nashville, TN 37232 (United States); Splittgerber, Ryan [Department of Cancer Biology, Vanderbilt University, School of Medicine, Nashville, TN 37232 (United States); Fan, Guo-Huang [Department of Neurobiology and Neurotoxicology, Meharry Medical College, Nashville, TN 37221 (United States); Richmond, Ann, E-mail: ann.richmond@vanderbilt.edu [VA Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University, School of Medicine, Nashville, TN 37232 (United States)

    2011-11-15

    Alzheimer's disease (AD) is characterized by a progressive cognitive decline and accumulation of neurotoxic oligomeric peptides amyloid-{beta} (A{beta}). Although the molecular events are not entirely known, it has become evident that inflammation, environmental and other risk factors may play a causal, disruptive and/or protective role in the development of AD. The present study investigated the ability of the chemokines, macrophage inflammatory protein-2 (MIP-2) and stromal cell-derived factor-1{alpha} (SDF-1{alpha}), the respective ligands for chemokine receptors CXCR2 and CXCR4, to suppress A{beta}-induced neurotoxicity in vitro and in vivo. Pretreatment with MIP-2 or SDF-1{alpha} significantly protected neurons from A{beta}-induced dendritic regression and apoptosis in vitro through activation of Akt, ERK1/2 and maintenance of metalloproteinase ADAM17 especially with SDF-1{alpha}. Intra-cerebroventricular (ICV) injection of A{beta} led to reduction in dendritic length and spine density of pyramidal neurons in the CA1 area of the hippocampus and increased oxidative damage 24 h following the exposure. The A{beta}-induced morphometric changes of neurons and increase in biomarkers of oxidative damage, F{sub 2}-isoprostanes, were significantly inhibited by pretreatment with the chemokines MIP-2 or SDF-1{alpha}. Additionally, MIP-2 or SDF-1{alpha} was able to suppress the aberrant mislocalization of p21-activated kinase (PAK), one of the proteins involved in the maintenance of dendritic spines. Furthermore, MIP-2 also protected neurons against A{beta} neurotoxicity in CXCR2-/- mice, potentially through observed up regulation of CXCR1 mRNA. Understanding the neuroprotective potential of chemokines is crucial in defining the role for their employment during the early stages of neurodegeneration. -- Research highlights: Black-Right-Pointing-Pointer Neuroprotective ability of the chemokines MIP2 and CXCL12 against A{beta} toxicity. Black-Right-Pointing-Pointer MIP

  17. Chemokines, macrophage inflammatory protein-2 and stromal cell-derived factor-1α, suppress amyloid β-induced neurotoxicity

    International Nuclear Information System (INIS)

    Raman, Dayanidhi; Milatovic, Snjezana-Zaja; Milatovic, Dejan; Splittgerber, Ryan; Fan, Guo-Huang; Richmond, Ann

    2011-01-01

    Alzheimer's disease (AD) is characterized by a progressive cognitive decline and accumulation of neurotoxic oligomeric peptides amyloid-β (Aβ). Although the molecular events are not entirely known, it has become evident that inflammation, environmental and other risk factors may play a causal, disruptive and/or protective role in the development of AD. The present study investigated the ability of the chemokines, macrophage inflammatory protein-2 (MIP-2) and stromal cell-derived factor-1α (SDF-1α), the respective ligands for chemokine receptors CXCR2 and CXCR4, to suppress Aβ-induced neurotoxicity in vitro and in vivo. Pretreatment with MIP-2 or SDF-1α significantly protected neurons from Aβ-induced dendritic regression and apoptosis in vitro through activation of Akt, ERK1/2 and maintenance of metalloproteinase ADAM17 especially with SDF-1α. Intra-cerebroventricular (ICV) injection of Aβ led to reduction in dendritic length and spine density of pyramidal neurons in the CA1 area of the hippocampus and increased oxidative damage 24 h following the exposure. The Aβ-induced morphometric changes of neurons and increase in biomarkers of oxidative damage, F 2 -isoprostanes, were significantly inhibited by pretreatment with the chemokines MIP-2 or SDF-1α. Additionally, MIP-2 or SDF-1α was able to suppress the aberrant mislocalization of p21-activated kinase (PAK), one of the proteins involved in the maintenance of dendritic spines. Furthermore, MIP-2 also protected neurons against Aβ neurotoxicity in CXCR2−/− mice, potentially through observed up regulation of CXCR1 mRNA. Understanding the neuroprotective potential of chemokines is crucial in defining the role for their employment during the early stages of neurodegeneration. -- Research highlights: ► Neuroprotective ability of the chemokines MIP2 and CXCL12 against Aβ toxicity. ► MIP-2 or CXCL12 prevented dendritic regression and apoptosis in vitro. ► Neuroprotection through activation of Akt, ERK

  18. Genotoxicity of 3-nitrobenzanthrone and 3-aminobenzanthrone in MutaMouse and lung epithelial cells derived from MutaMouse.

    Science.gov (United States)

    Arlt, Volker M; Gingerich, John; Schmeiser, Heinz H; Phillips, David H; Douglas, George R; White, Paul A

    2008-11-01

    FE1 lung epithelial cells derived from MutaMouse are a new model system to provide in vitro mutagenicity data with the potential to predict the outcome of an in vivo MutaMouse test. 3-Nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust and urban air pollution. We investigated the mutagenicity and DNA binding of 3-NBA and its main metabolite 3-aminobenzanthrone (3-ABA) in vitro and in vivo in the MutaMouse assay. Mice were treated with 3-NBA or 3-ABA (0, 2 or 5 mg/kg body weight/day) by gavage for 28 days and 28 days later lacZ mutant frequency (MF) was determined in liver, lung and bone marrow. For both compounds, dose-related increases in MF were seen in liver and bone marrow, but not in lung; mutagenic activity was approximately 2-fold lower for 3-ABA than for 3-NBA. With 3-NBA, highest DNA adduct levels (measured by (32)P-post-labelling) were found in liver (approximately 230 adducts per 10(8) nucleotides) with levels 20- to 40-fold lower in bone marrow and lung. With 3-ABA, DNA adduct levels were again highest in the liver, but approximately 4-fold lower than for 3-NBA. FE1 cells were exposed to up to 10 microg/ml 3-NBA or 3-ABA for 6 h with or without exogenous activation (S9) and harvested after 3 days. For 3-NBA, there was a dose-related increase in MF both with and without S9 mix, which was >10 times higher than observed in vivo. At the highest concentration of 3-ABA (10 microg/ml), we found only around a 2-fold increase in MF relative to controls. DNA adduct formation in FE1 cells was dose-dependent for both compounds, but 10- to 20-fold higher for 3-NBA compared to 3-ABA. Collectively, our data indicate that MutaMouse FE1 cells are well suited for cost-effective testing of suspected mutagens with different metabolic activation pathways as a guide for subsequent in vivo MutaMouse testing.

  19. In vitro cardiotoxicity assessment of environmental chemicals using an organotypic human induced pluripotent stem cell-derived model

    Energy Technology Data Exchange (ETDEWEB)

    Sirenko, Oksana, E-mail: oksana.sirenko@moldev.com [Molecular Devices, LLC, Sunnyvale, CA (United States); Grimm, Fabian A. [Department of Veterinary Integrative Biosciences, Texas A& M University, College Station, TX (United States); Ryan, Kristen R. [Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC (United States); Iwata, Yasuhiro; Chiu, Weihsueh A. [Department of Veterinary Integrative Biosciences, Texas A& M University, College Station, TX (United States); Parham, Frederick [Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC (United States); Wignall, Jessica A. [ICF, Fairfax, VA (United States); Anson, Blake [Cellular Dynamics International, Madison, WI (United States); Cromwell, Evan F. [Protein Fluidics, Inc., Burlingame, CA (United States); Behl, Mamta [Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC (United States); Rusyn, Ivan [Department of Veterinary Integrative Biosciences, Texas A& M University, College Station, TX (United States); Tice, Raymond R. [Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC (United States)

    2017-05-01

    An important target area for addressing data gaps through in vitro screening is the detection of potential cardiotoxicants. Despite the fact that current conservative estimates relate at least 23% of all cardiovascular disease cases to environmental exposures, the identities of the causative agents remain largely uncharacterized. Here, we evaluate the feasibility of a combinatorial in vitro/in silico screening approach for functional and mechanistic cardiotoxicity profiling of environmental hazards using a library of 69 representative environmental chemicals and drugs. Human induced pluripotent stem cell-derived cardiomyocytes were exposed in concentration-response for 30 min or 24 h and effects on cardiomyocyte beating and cellular and mitochondrial toxicity were assessed by kinetic measurements of intracellular Ca{sup 2+} flux and high-content imaging using the nuclear dye Hoechst 33342, the cell viability marker Calcein AM, and the mitochondrial depolarization probe JC-10. More than half of the tested chemicals exhibited effects on cardiomyocyte beating after 30 min of exposure. In contrast, after 24 h, effects on cell beating without concomitant cytotoxicity were observed in about one third of the compounds. Concentration-response data for in vitro bioactivity phenotypes visualized using the Toxicological Prioritization Index (ToxPi) showed chemical class-specific clustering of environmental chemicals, including pesticides, flame retardants, and polycyclic aromatic hydrocarbons. For environmental chemicals with human exposure predictions, the activity-to-exposure ratios between modeled blood concentrations and in vitro bioactivity were between one and five orders of magnitude. These findings not only demonstrate that some ubiquitous environmental pollutants might have the potential at high exposure levels to alter cardiomyocyte function, but also indicate similarities in the mechanism of these effects both within and among chemicals and classes. - Highlights:

  20. Ca2+-currents in human induced pluripotent stem cell-derived cardiomyocytes - effects of two different culture conditions

    Directory of Open Access Journals (Sweden)

    Ahmet Umur Uzun

    2016-09-01

    Full Text Available Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM provide a unique opportunity to study human heart physiology and pharmacology and repair injured hearts. The suitability of hiPSC-CM critically depends on how closely they share physiological properties of human adult cardiomyocytes (CM. Here we investigated whether a 3D engineered heart tissue (EHT culture format favors maturation and addressed the L-type Ca2+-current (ICa,L as a readout. The results were compared with hiPSC-CM cultured in conventional monolayer (ML and to our previous data from human adult atrial and ventricular CM obtained when identical patch-clamp protocols were used. HiPSC-CM were 2-3 fold smaller than adult CM, independently of culture format (capacitance ML 45±1 pF (n=289, EHT 45±1 pF (n=460, atrial CM 87±3 pF (n=196, ventricular CM 126±8 pF (n=50. Only 88% of ML cells showed ICa, but all EHT. Basal ICa density was 10±1 pA/pF (n=207 for ML and 12±1 pA/pF (n=361 for EHT and was larger than in adult CM (7±1 pA/pF (p<0.05, n=196 for atrial CM and 6±1 pA/pF (p<0.05, n=47 for ventricular CM. However, ML and EHT showed robust T-type Ca2+-currents (ICa,T. While (--Bay K 8644, that activates ICa,L directly, increased ICa,L to the same extent in ML and EHT, β1- and β2-adrenoceptor effects were marginal in ML, but of same size as (--Bay K 8644 in EHT. The opposite was true for serotonin receptors. Sensitivity to β1 and β2-adrenoceptor stimulation was the same in EHT as in adult CM (-logEC50: 5.9 and 6.1 for norepinephrine (NE and epinephrine (Epi, respectively, but very low concentrations of Rp-8-Br-cAMPS were sufficient to suppress effects (-logEC50: 5.3 and 5.3 respectively for NE and Epi. Taken together, hiPSC-CM express ICa,L at the same density as human adult CM, but, in contrast, possess robust ICa,T. Increased effects of catecholamines in EHT suggest more efficient maturation.

  1. Prolyl hydroxylation regulates protein degradation, synthesis, and splicing in human induced pluripotent stem cell-derived cardiomyocytes.

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    Stoehr, Andrea; Yang, Yanqin; Patel, Sajni; Evangelista, Alicia M; Aponte, Angel; Wang, Guanghui; Liu, Poching; Boylston, Jennifer; Kloner, Philip H; Lin, Yongshun; Gucek, Marjan; Zhu, Jun; Murphy, Elizabeth

    2016-06-01

    Protein hydroxylases are oxygen- and α-ketoglutarate-dependent enzymes that catalyse hydroxylation of amino acids such as proline, thus linking oxygen and metabolism to enzymatic activity. Prolyl hydroxylation is a dynamic post-translational modification that regulates protein stability and protein-protein interactions; however, the extent of this modification is largely uncharacterized. The goals of this study are to investigate the biological consequences of prolyl hydroxylation and to identify new targets that undergo prolyl hydroxylation in human cardiomyocytes. We used human induced pluripotent stem cell-derived cardiomyocytes in combination with pulse-chase amino acid labelling and proteomics to analyse the effects of prolyl hydroxylation on protein degradation and synthesis. We identified 167 proteins that exhibit differences in degradation with inhibition of prolyl hydroxylation by dimethyloxalylglycine (DMOG); 164 were stabilized. Proteins involved in RNA splicing such as serine/arginine-rich splicing factor 2 (SRSF2) and splicing factor and proline- and glutamine-rich (SFPQ) were stabilized with DMOG. DMOG also decreased protein translation of cytoskeletal and sarcomeric proteins such as α-cardiac actin. We searched the mass spectrometry data for proline hydroxylation and identified 134 high confidence peptides mapping to 78 unique proteins. We identified SRSF2, SFPQ, α-cardiac actin, and cardiac titin as prolyl hydroxylated. We identified 29 prolyl hydroxylated proteins that showed a significant difference in either protein degradation or synthesis. Additionally, we performed next-generation RNA sequencing and showed that the observed decrease in protein synthesis was not due to changes in mRNA levels. Because RNA splicing factors were prolyl hydroxylated, we investigated splicing ± inhibition of prolyl hydroxylation and detected 369 alternative splicing events, with a preponderance of exon skipping. This study provides the first extensive

  2. Epigenetic regulation of cardiac progenitor cells marker c-kit by stromal cell derived factor-1α.

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    Zhongpu Chen

    Full Text Available BACKGROUND: Cardiac progenitor cells (CPCs have been proven suitable for stem cell therapy after myocardial infarction, especially c-kit(+CPCs. CPCs marker c-kit and its ligand, the stem cell factor (SCF, are linked as c-kit/SCF axis, which is associated with the functions of proliferation and differentiation. In our previous study, we found that stromal cell-derived factor-1α (SDF-1α could enhance the expression of c-kit. However, the mechanism is unknown. METHODS AND RESULTS: CPCs were isolated from adult mouse hearts, c-kit(+ and c-kit(- CPCs were separated by magnetic beads. The cells were cultured with SDF-1α and CXCR4-selective antagonist AMD3100, and c-kit expression was measured by qPCR and Western blotting. Results showed that SDF-1α could enhance c-kit expression of c-kit(+CPCs, made c-kit(-CPCs expressing c-kit, and AMD3100 could inhibit the function of SDF-1α. After the intervention of SDF-1α and AMD3100, proliferation and migration of CPCs were measured by CCK-8 and transwell assay. Results showed that SDF-1α could enhance the proliferation and migration of both c-kit(+ and c-kit(- CPCs, and AMD3100 could inhibit these functions. DNA methyltransferase (DNMT mRNA were measured by qPCR, DNMT activity was measured using the DNMT activity assay kit, and DNA methylation was analyzed using Sequenom's MassARRAY platform, after the CPCs were cultured with SDF-1α. The results showed that SDF-1α stimulation inhibited the expression of DNMT1 and DNMT3β, which are critical for the maintenance of regional DNA methylation. Global DNMT activity was also inhibited by SDF-1α. Lastly, SDF-1α treatment led to significant demethylation in both c-kit(+ and c-kit(- CPCs. CONCLUSIONS: SDF-1α combined with CXCR4 could up-regulate c-kit expression of c-kit(+CPCs and make c-kit(-CPCs expressing c-kit, which result in the CPCs proliferation and migration ability improvement, through the inhibition of DNMT1 and DNMT3β expression and global DNMT

  3. Characterization of transcription factor networks involved in umbilical cord blood CD34+ stem cells-derived erythropoiesis.

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    Biaoru Li

    Full Text Available Fetal stem cells isolated from umbilical cord blood (UCB possess a great capacity for proliferation and differentiation and serve as a valuable model system to study gene regulation. Expanded knowledge of the molecular control of hemoglobin synthesis will provide a basis for rational design of therapies for β-hemoglobinopathies. Transcriptome data are available for erythroid progenitors derived from adult stem cells, however studies to define molecular mechanisms controlling globin gene regulation during fetal erythropoiesis are limited. Here, we utilize UCB-CD34+ stem cells induced to undergo erythroid differentiation to characterize the transcriptome and transcription factor networks (TFNs associated with the γ/β-globin switch during fetal erythropoiesis. UCB-CD34+ stem cells grown in the one-phase liquid culture system displayed a higher proliferative capacity than adult CD34+ stem cells. The γ/β-globin switch was observed after day 42 during fetal erythropoiesis in contrast to adult progenitors where the switch occurred around day 21. To gain insights into transcription factors involved in globin gene regulation, microarray analysis was performed on RNA isolated from UCB-CD34+ cell-derived erythroid progenitors harvested on day 21, 42, 49 and 56 using the HumanHT-12 Expression BeadChip. After data normalization, Gene Set Enrichment Analysis identified transcription factors (TFs with significant changes in expression during the γ/β-globin switch. Forty-five TFs were silenced by day 56 (Profile-1 and 30 TFs were activated by day 56 (Profile-2. Both GSEA datasets were analyzed using the MIMI Cytoscape platform, which discovered TFNs centered on KLF4 and GATA2 (Profile-1 and KLF1 and GATA1 for Profile-2 genes. Subsequent shRNA studies in KU812 leukemia cells and human erythroid progenitors generated from UCB-CD34+ cells supported a negative role of MAFB in γ-globin regulation. The characteristics of erythroblasts derived from UCB-CD34

  4. Regulation of alternative macrophage activation in the liver following acetaminophen intoxication by stem cell-derived tyrosine kinase

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    Gardner, Carol R., E-mail: cgardner@pharmacy.rutgers.edu [Department of Pharmacology and Toxicology, Rutgers University, Ernest Mario School of Pharmacy, Piscataway, NJ 08854 (United States); Hankey, Pamela [Department of Veterinary and Biomedical Science, Pennsylvania State University, University Park, PA 16802 (United States); Mishin, Vladimir; Francis, Mary [Department of Pharmacology and Toxicology, Rutgers University, Ernest Mario School of Pharmacy, Piscataway, NJ 08854 (United States); Yu, Shan [Department of Veterinary and Biomedical Science, Pennsylvania State University, University Park, PA 16802 (United States); Laskin, Jeffrey D. [Department of Environmental and Occupational Medicine, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854 (United States); Laskin, Debra L. [Department of Pharmacology and Toxicology, Rutgers University, Ernest Mario School of Pharmacy, Piscataway, NJ 08854 (United States)

    2012-07-15

    Stem cell-derived tyrosine kinase (STK) is a transmembrane receptor reported to play a role in macrophage switching from a classically activated/proinflammatory phenotype to an alternatively activated/wound repair phenotype. In the present studies, STK{sup −/−} mice were used to assess the role of STK in acetaminophen-induced hepatotoxicity as evidence suggests that the pathogenic process involves both of these macrophage subpopulations. In wild type mice, centrilobular hepatic necrosis and increases in serum transaminase levels were observed within 6 h of acetaminophen administration (300 mg/kg, i.p.). Loss of STK resulted in a significant increase in sensitivity of mice to the hepatotoxic effects of acetaminophen and increased mortality, effects independent of its metabolism. This was associated with reduced levels of hepatic glutathione, rapid upregulation of inducible nitric oxide synthase, and prolonged induction of heme oxygenase-1, suggesting excessive oxidative stress in STK{sup −/−} mice. F4/80, a marker of mature macrophages, was highly expressed on subpopulations of Kupffer cells in livers of wild type, but not STK{sup −/−} mice. Whereas F4/80{sup +} macrophages rapidly declined in the livers of wild type mice following acetaminophen intoxication, they increased in STK{sup −/−} mice. In wild type mice hepatic expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-12, products of classically activated macrophages, increased after acetaminophen administration. Monocyte chemotactic protein-1 (MCP-1) and its receptor, CCR2, as well as IL-10, mediators involved in recruiting and activating anti-inflammatory/wound repair macrophages, also increased in wild type mice after acetaminophen. Loss of STK blunted the effects of acetaminophen on expression of TNFα, IL-1β, IL-12, MCP-1 and CCR2, while expression of IL-10 increased. Hepatic expression of CX3CL1, and its receptor, CX3CR1 also increased in STK{sup −/−} mice

  5. In vitro cardiotoxicity assessment of environmental chemicals using an organotypic human induced pluripotent stem cell-derived model

    International Nuclear Information System (INIS)

    Sirenko, Oksana; Grimm, Fabian A.; Ryan, Kristen R.; Iwata, Yasuhiro; Chiu, Weihsueh A.; Parham, Frederick; Wignall, Jessica A.; Anson, Blake; Cromwell, Evan F.; Behl, Mamta; Rusyn, Ivan; Tice, Raymond R.

    2017-01-01

    An important target area for addressing data gaps through in vitro screening is the detection of potential cardiotoxicants. Despite the fact that current conservative estimates relate at least 23% of all cardiovascular disease cases to environmental exposures, the identities of the causative agents remain largely uncharacterized. Here, we evaluate the feasibility of a combinatorial in vitro/in silico screening approach for functional and mechanistic cardiotoxicity profiling of environmental hazards using a library of 69 representative environmental chemicals and drugs. Human induced pluripotent stem cell-derived cardiomyocytes were exposed in concentration-response for 30 min or 24 h and effects on cardiomyocyte beating and cellular and mitochondrial toxicity were assessed by kinetic measurements of intracellular Ca 2+ flux and high-content imaging using the nuclear dye Hoechst 33342, the cell viability marker Calcein AM, and the mitochondrial depolarization probe JC-10. More than half of the tested chemicals exhibited effects on cardiomyocyte beating after 30 min of exposure. In contrast, after 24 h, effects on cell beating without concomitant cytotoxicity were observed in about one third of the compounds. Concentration-response data for in vitro bioactivity phenotypes visualized using the Toxicological Prioritization Index (ToxPi) showed chemical class-specific clustering of environmental chemicals, including pesticides, flame retardants, and polycyclic aromatic hydrocarbons. For environmental chemicals with human exposure predictions, the activity-to-exposure ratios between modeled blood concentrations and in vitro bioactivity were between one and five orders of magnitude. These findings not only demonstrate that some ubiquitous environmental pollutants might have the potential at high exposure levels to alter cardiomyocyte function, but also indicate similarities in the mechanism of these effects both within and among chemicals and classes. - Highlights:

  6. Age Is Relative—Impact of Donor Age on Induced Pluripotent Stem Cell-Derived Cell Functionality

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    Elisabeth Tamara Strässler

    2018-01-01

    Full Text Available Induced pluripotent stem cells (iPSCs avoid many of the restrictions that hamper the application of human embryonic stem cells: limited availability of source material due to legal restrictions in some countries, immunogenic rejection and ethical concerns. Also, the donor’s clinical phenotype is often known when working with iPSCs. Therefore, iPSCs seem ideal to tackle the two biggest tasks of regenerative medicine: degenerative diseases with genetic cause (e.g., Duchenne’s muscular dystrophy and organ replacement in age-related diseases (e.g., end-stage heart or renal failure, especially in combination with recently developed gene-editing tools. In the setting of autologous transplantation in elderly patients, donor age becomes a potentially relevant factor that needs to be assessed. Here, we review and critically discuss available data pertinent to the questions: How does donor age influence the reprogramming process and iPSC functionality? Would it even be possible to reprogram senescent somatic cells? How does donor age affect iPSC differentiation into specialised cells and their functionality? We also identify research needs, which might help resolve current unknowns. Until recently, most hallmarks of ageing were attributed to an accumulation of DNA damage over time, and it was thus expected that DNA damage from a somatic cell would accumulate in iPSCs and the cells derived from them. In line with this, a decreased lifespan of cloned organisms compared with the donor was also observed in early cloning experiments. Therefore, it was questioned for a time whether iPSC derived from an old individual’s somatic cells would suffer from early senescence and, thus, may not be a viable option either for disease modelling nor future clinical applications. Instead, typical signs of cellular ageing are reverted in the process of iPSC reprogramming, and iPSCs from older donors do not show diminished differentiation potential nor do i

  7. Examining the protective role of ErbB2 modulation in human-induced pluripotent stem cell-derived cardiomyocytes.

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    Eldridge, Sandy; Guo, Liang; Mussio, Jodie; Furniss, Mike; Hamre, John; Davis, Myrtle

    2014-10-01

    Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are being used as an in vitro model system in cardiac biology and in drug discovery (e.g., cardiotoxicity testing). Qualification of these cells for use in mechanistic investigations will require detailed evaluations of cardiomyocyte signaling pathways and cellular responses. ErbB signaling and the ligand neuregulin play critical roles in survival and functional integrity of cardiac myocytes. As such, we sought to characterize the expression and activity of the ErbB family of receptors. Antibody microarray analysis performed on cell lysates derived from maturing hiPSC-CMs detected expression of ∼570 signaling proteins. EGFR/ErbB1, HER2/ErbB2, and ErbB4, but not ErbB3 receptors, of the epidermal growth factor receptor family were confirmed by Western blot. Activation of ErbB signaling by neuregulin-1β (NRG, a natural ligand for ErbB4) and its modulation by trastuzumab (a monoclonal anti-ErbB2 antibody) and lapatinib (a small molecule ErbB2 tyrosine kinase inhibitor) were evaluated through assessing phosphorylation of AKT and Erk1/2, two major downstream kinases of ErbB signaling, using nanofluidic proteomic immunoassay. Downregulation of ErbB2 expression by siRNA silencing attenuated NRG-induced AKT and Erk1/2 phosphorylation. Activation of ErbB signaling with NRG, or inhibition with trastuzumab, alleviated or aggravated doxorubicin-induced cardiomyocyte damage, respectively, as assessed by a real-time cellular impedance analysis and ATP measurement. Collectively, these results support the expanded use of hiPSC-CMs to examine mechanisms of cardiotoxicity and support the value of using these cells in early assessments of cardiotoxicity or efficacy. Published by Oxford University Press on behalf of Toxicological Sciences 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  8. Using the cost-effectiveness of allogeneic islet transplantation to inform induced pluripotent stem cell-derived β-cell therapy reimbursement.

    Science.gov (United States)

    Archibald, Peter R T; Williams, David J

    2015-11-01

    In the present study a cost-effectiveness analysis of allogeneic islet transplantation was performed and the financial feasibility of a human induced pluripotent stem cell-derived β-cell therapy was explored. Previously published cost and health benefit data for islet transplantation were utilized to perform the cost-effectiveness and sensitivity analyses. It was determined that, over a 9-year time horizon, islet transplantation would become cost saving and 'dominate' the comparator. Over a 20-year time horizon, islet transplantation would incur significant cost savings over the comparator (GB£59,000). Finally, assuming a similar cost of goods to islet transplantation and a lack of requirement for immunosuppression, a human induced pluripotent stem cell-derived β-cell therapy would dominate the comparator over an 8-year time horizon.

  9. Cancer Cell-derived Exosomes Induce Mitogen-activated Protein Kinase-dependent Monocyte Survival by Transport of Functional Receptor Tyrosine Kinases*

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    Song, Xiao; Ding, Yanping; Liu, Gang; Yang, Xiao; Zhao, Ruifang; Zhang, Yinlong; Zhao, Xiao; Anderson, Gregory J.; Nie, Guangjun

    2016-01-01

    Tumor-associated macrophages (TAM) play pivotal roles in cancer initiation and progression. Monocytes, the precursors of TAMs, normally undergo spontaneous apoptosis within 2 days, but can subsist in the inflammatory tumor microenvironment for continuous survival and generation of sufficient TAMs. The mechanisms underlying tumor-driving monocyte survival remain obscure. Here we report that cancer cell-derived exosomes were crucial mediators for monocyte survival in the inflammatory niche. Analysis of the survival-promoting molecules in monocytes revealed that cancer cell-derived exosomes activated Ras and extracellular signal-regulated kinases in the mitogen-activated protein kinase (MAPK) pathway, resulting in the prevention of caspase cleavage. Phosphorylated receptor tyrosine kinases (RTKs), such as phosphorylated epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2), were abundantly expressed in cancer cell-derived exosomes. Knock-out of EGFR or/and HER-2, or alternatively, inhibitors against their phosphorylation significantly disturbed the exosome-mediated activation of the MAPK pathway, inhibition of caspase cleavage, and increase in survival rate in monocytes. Moreover, the deprived survival-stimulating activity of exosomes due to null expression of EGFR and HER-2 could be restored by activation of another RTK, insulin receptor. Overall, our study uncovered a mechanism of tumor-associated monocyte survival and demonstrated that cancer cell-derived exosomes can stimulate the MAPK pathway in monocytes through transport of functional RTKs, leading to inactivation of apoptosis-related caspases. This work provides insights into the long sought question on monocyte survival prior to formation of plentiful TAMs in the tumor microenvironment. PMID:26895960

  10. Cancer Cell-derived Exosomes Induce Mitogen-activated Protein Kinase-dependent Monocyte Sur