The Experts below are selected from a list of 975 Experts worldwide ranked by ideXlab platform
Alan F Murray - One of the best experts on this subject based on the ideXlab platform.
-
Video Article Cell Patterning on Photolithographically Defined Parylene-C: SiO2 Substrates
2016Co-Authors: Mark A. Hughes, Paul M. Brennan, Andrew S. Bunting, Mike J. Shipston, Alan F MurrayAbstract:Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of Cellular physiology. To easily combine Cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based ‘lab on a chip ’ technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2 wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured Cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of Cell types (including primary murine hippocampal Cells, HEK 293 Cell Line, human neuron-like Teratocarcinoma Cell Line, primary murine cerebellar granule Cells, and primary human glioma-derived stem-like Cells). Interestingly, however, the platform is not universal; reflecting the importance of Cell-specific adhesion molecules
-
First human hNT astrocytes patterned to single Cell resolution on parylene-C/Silicon dioxide substrates
2011 Annual International Conference of the IEEE Engineering in Medicine and Biology Society, 2011Co-Authors: Charles P Unsworth, Evangelos Delivopoulos, Euan S. Graham, Alan F MurrayAbstract:In our previous work we developed a successful protocol to pattern the human hNT neuron (derived from the human Teratocarcinoma Cell Line (hNT)) on parylene-C/SiO2 substrates. This communication, reports how we have successfully managed to pattern the supportive Cell to the neuron, the hNT astrocyte, on such substrates. Here we disseminate the nanofabrication, Cell differentiation and Cell culturing protocols necessary to successfully pattern the first human hNT astrocytes to single Cell resolution on parylene-C/SiO2 substrates. This is performed for varying parylene strip widths providing exCellent contrast to the SiO2 substrate and elegant single Cell isolation at 10μm strip widths. The breakthrough in patterning human Cells on a silicon chip has widespread implications and is valuable as a platform technology as it enables a detailed study of the human brain at the Cellular and network level.
-
first human hnt neurons patterned on parylene c silicon dioxide substrates combining an accessible Cell Line and robust patterning technology for the study of the pathological adult human brain
Journal of Neuroscience Methods, 2010Co-Authors: Charles P Unsworth, E Graham, Evangelos Delivopoulos, Mike Dragunow, Alan F MurrayAbstract:In this communication, we describe a new method which has enabled the first patterning of human neurons (derived from the human Teratocarcinoma Cell Line (hNT)) on parylene-C/silicon dioxide substrates. We reveal the details of the nanofabrication processes, Cell differentiation and culturing protocols necessary to successfully pattern hNT neurons which are each key aspects of this new method. The benefits in patterning human neurons on silicon chip using an accessible Cell Line and robust patterning technology are of widespread value. Thus, using a combined technology such as this will facilitate the detailed study of the pathological human brain at both the single Cell and network level.
John Q. Trojanowski - One of the best experts on this subject based on the ideXlab platform.
-
transplanted human neurons derived from a Teratocarcinoma Cell Line ntera 2 mature integrate and survive for over 1 year in the nude mouse brain
The Journal of Comparative Neurology, 1995Co-Authors: Sofie R Kleppner, John Q. Trojanowski, Kathryn A Robinson, Virginia M Y LeeAbstract:Retinoic acid (RA) induces a human Teratocarcinoma Cell Line (NTera-2 or NT2) to give rise exclusively to post-mitotic neuron-like (NT2N) Cells, but NT2N Cells never acquire a fully mature neuronal phenotype in vitro. To determine whether NT2N Cells can mature into adult neuron-like Cells in vivo, purified NT2N Cells were grafted into different regions of the central nervous system (CNS) of adult and neonatal athymic mice, and the grafts were examined immunohistochemically by light, confocal, and electron microscopy using antibodies to a panel of developmentally regulated neuronal polypeptides. NT2N grafts were distinguished from endogenous mouse neurons with antibodies that recognize human or murine specific epitopes in selected neuronal polypeptides. Viable NT2N Cells were identified in > 89% of graft recipients (N = 90), and some grafts survived 14 months. Within 3 weeks of implantation, grafted NT2N Cells re-extended their processes, and the location of the grafts (e.g., septum versus neocortex) appeared to determine the extent to which processes were elaborated. Within the early post-transplantation period, grafted NT2N Cells expressed the same neuronal polypeptides as their in vitro counterparts. However, between 6 weeks and 4-6 months post-implantation, the grafted NT2N Cells progressively acquired the molecular phenotype of fully mature in vivo neurons as evidenced by dramatically increased expression of the most highly phosphorylated isoforms of the heavy neurofilament subunit, and the de novo expression of adult CNS tau. Notably, the time course for the extension of processes and the expression of neuronal polypeptides by NT2N grafts was similar in neonatal and adult mice. Although grafted NT2N Cells formed synapse-like structures and elaborated dendrites and axons, these axons remained unmyelinated. Finally, none of the transplanted NT2N Cells reverted to a neoplastic state. These studies demonstrate that pure populations of grafted human NT2N Cells acquire a fully mature neuronal phenotype in vivo, and that these Cells integrate and survive for > 1 year post-implantation in the mouse CNS. These human neuron-like Cells are an attractive model system for studies of neuronal development, polarity and transplantation.
-
human neurons derived from a Teratocarcinoma Cell Line express solely the 695 amino acid amyloid precursor protein and produce intraCellular beta amyloid or a4 peptides
Proceedings of the National Academy of Sciences of the United States of America, 1993Co-Authors: Andrew M Wertkin, Todd E. Golde, Samuel J Pleasure, Steven G Younkin, R.s. Turner, John Q. TrojanowskiAbstract:The beta-amyloid or beta/A4 peptides that accumulate as filamentous aggregates in the extraCellular space of Alzheimer disease (AD) brains are derived from one or more alternatively spliced amyloid precursor proteins (APPs). The more abundant APPs in the central nervous system are the 695-(APP695), 751- (APP751), and 770- (APP770) amino acid isoforms, and each could be the source of beta/A4 peptide that accumulates in the AD brain. It is plausible that altered metabolism of these APPs by central nervous system neurons could lead to the release and deposition of beta/A4 peptide in brain parenchyma. Thus, we examined the expression and processing of the three major brain APPs in nearly pure human neurons (NT2N Cells) derived from a Teratocarcinoma Cell Line (NTera2/c1.D1 or NT2 Cells) after retinoic acid treatment. NT2N neurons expressed almost exclusively APP695, whereas NT2 Cells expressed predominantly APP751/770. Furthermore, the processing of the APPs in NT2N Cells was distinct from NT2 and nonneuronal Cells. Most significantly, the NT2N neurons but not the NT2 Cells constitutively generated intraCellular beta/A4 peptide and released it into the culture medium. This work demonstrates the intraCellular production of beta/A4 peptide and suggests that cultured NT2N Cells may provide a unique model system for understanding the contribution of neurons and APP695 to amyloidogenesis in the AD brain.
-
neurons derived from a human Teratocarcinoma Cell Line establish molecular and structural polarity following transplantation into the rodent brain
Experimental Neurology, 1993Co-Authors: John Q. Trojanowski, John R Mantione, Derice P Seid, Landon J IngeAbstract:Abstract Studies of neurons grafted into the brains of experimental animals have been limited by the lack of suitably homogeneous populations of neurons for transplantation. Here we describe the transplantation and survival of pure, postmitotic human neurons (NT2N Cells) into the rat brain. NT2N Cells were derived from a human Teratocarcinoma Line (NTera2/clone D1 or NT2 Cells) in vitro by retinoic acid treatment. Approximately 5-10 × 104 NT2N Cells (including previously frozen aliquots of NT2N Cells) were injected into the neocortex, subjacent white matter, or hippocampus of adult (N = 51) or neonatal (N = 17) Sprague-Dawley rats. Cyclosporine (7-10 mg/kg) was administered daily to 13 adult rats for up to 12 weeks post-transplant prior to sacrifice. Untreated rats survived for up to 21 weeks post-transplant. Injection sites were serially sectioned and NT2N grafts were analyzed immunohistochemically using antibodies to diverse neuronal and glial proteins to assess the Lineage of the grafted Cells and their ability to establish molecular and structural polarity. NT2N Cells transplanted into untreated adult and neonatal rat brains were committed exclusively to the neuronal phenotype and survived for as long as 8 weeks, although most were rejected after 4 weeks. However, cyclosporine prolonged survival of the NT2N grafts for up to 12 weeks. Further, grafted NT2N Cells exhibited an asymmetric geometry (with long axons and simplified dendrites), as well as molecular polarity (with highly phosphorylated neurofilament proteins segregated in axons and microtubule associated protein 2 confined to perikarya and dendrites) by 4 weeks post-transplant. However, the grafted neurons did not become fully mature as evidenced by their failure to express the most highly phosphorylated heavy neurofilament proteins. Finally, previously frozen NT2N Cells survived in the rat brain, and none of the grafts formed neoplasms. We conclude from these studies that transplanted NT2N Cells represent a highly advantageous model system for studies of the developmental biology of neurons.
Charles P Unsworth - One of the best experts on this subject based on the ideXlab platform.
-
First human hNT astrocytes patterned to single Cell resolution on parylene-C/Silicon dioxide substrates
2011 Annual International Conference of the IEEE Engineering in Medicine and Biology Society, 2011Co-Authors: Charles P Unsworth, Evangelos Delivopoulos, Euan S. Graham, Alan F MurrayAbstract:In our previous work we developed a successful protocol to pattern the human hNT neuron (derived from the human Teratocarcinoma Cell Line (hNT)) on parylene-C/SiO2 substrates. This communication, reports how we have successfully managed to pattern the supportive Cell to the neuron, the hNT astrocyte, on such substrates. Here we disseminate the nanofabrication, Cell differentiation and Cell culturing protocols necessary to successfully pattern the first human hNT astrocytes to single Cell resolution on parylene-C/SiO2 substrates. This is performed for varying parylene strip widths providing exCellent contrast to the SiO2 substrate and elegant single Cell isolation at 10μm strip widths. The breakthrough in patterning human Cells on a silicon chip has widespread implications and is valuable as a platform technology as it enables a detailed study of the human brain at the Cellular and network level.
-
first human hnt neurons patterned on parylene c silicon dioxide substrates combining an accessible Cell Line and robust patterning technology for the study of the pathological adult human brain
Journal of Neuroscience Methods, 2010Co-Authors: Charles P Unsworth, E Graham, Evangelos Delivopoulos, Mike Dragunow, Alan F MurrayAbstract:In this communication, we describe a new method which has enabled the first patterning of human neurons (derived from the human Teratocarcinoma Cell Line (hNT)) on parylene-C/silicon dioxide substrates. We reveal the details of the nanofabrication processes, Cell differentiation and culturing protocols necessary to successfully pattern hNT neurons which are each key aspects of this new method. The benefits in patterning human neurons on silicon chip using an accessible Cell Line and robust patterning technology are of widespread value. Thus, using a combined technology such as this will facilitate the detailed study of the pathological human brain at both the single Cell and network level.
Büscher Kristina - One of the best experts on this subject based on the ideXlab platform.
-
Expression und biologische Funktion von humanen endogenen Retroviren (HERVs), insbesondere von HERV-K
Robert Koch-Institut, 2006Co-Authors: Büscher KristinaAbstract:Daten des humanen Genomprojektes zeigen, dass ca. 8% des gesamten humanen Genoms aus retroviralen Sequenzen besteht. Der überwiegende Teil dieser Proviren ist aufgrund verschiedener Mutationen defekt. Im Gegensatz zu allen anderen HERV Proviren scheinen einige HERV-K Proviren intakt zu sein und besitzen offene Leserahmen für alle viralen Proteine. Die Familie des humanen endogenen Retrovirus K HML2 umfasst ca. 30 eng verwandte Proviren. Zusätzlich zu den Strukturproteinen Gag und Env und der Reversen Transkriptase, exprimiert HERV-K zwei regulatorische Proteine, Rec und Np9. Beide sind im Nukleus lokalisiert und tumorigene Eigenschaften bzw. eine Expression in Assoziation mit Tumorgeweben wurde nachgewiesen. Neben Zelllinien, wie die Teratokarzinomzelllinie GH und einigen Brustkrebszelllinien, für die die Expression von HERV-K mRNA und die Produktion von Viruspartikeln bekannt ist, konnte die Expression von HERV-K Proteinen und Partikeln für Melanomzellen gezeigt werden. Volllängen mRNA von HERV-K war in allen untersuchten humanen Proben nachweisbar. Gespleißtes env und rec war in 39% der Gewebe und in 38% der Melanomzelllinien exprimiert. Zusätzlich werden HERV-H, -R und -W exprimiert. Von den auf spezifische Antikörper gegen HERV-K Proteine untersuchten Seren der Melanompatienten waren 16% positiv für das transmembrane Hüllprotein, jedoch reagierte kein Serum mit Rec oder Np9. Da im Zuge der Entstehung von Tumoren immer auch eine Dedifferenzierung der entarteten Zellen diskutiert wird, wurde die Expression von HERVs in undifferenzierten, embryonalen Stammzellen bestimmt. In den untersuchten embryonalen Stammzellen lässt sich Volllängen mRNA, sowie gespleißte env, rec und np9 mRNA nachweisen. Während der Differenzierung zu neuronalen Vorläuferzellen sinkt die Expression jedoch wieder auf ein mit normalen Zellen vergleichbares Niveau. Obwohl gespleißte RNA und virale Proteine von HERV-K vor allem in Tumoren und Tumorzelllinien exprimiert werden, ist deren Funktion während der Tumorentstehung noch immer ungeklärt. Auch die Bedeutung der HERV-K Expression in humanen Stammzellen ist noch unklar, insbesondere in Hinblick auf eine mögliche Tumorigenität.In contrast to all other human endogenous retroviruses, proviruses of the human endogenous retrovirus family HERV-K have maintained open reading frames for all viral proteins. Although most proviruses are defective, structural proteins Gag and Env, the reverse transcriptase and two regulatory proteins, Rec and Np9, have been described. Rec resembles the Rev protein of HIV and tumourigenic potential was confirmed. Np9 as well is located in the nucleus and expression in association with tumour tissues was observed. Additionally to Cell Lines known to produce HERV-K virus particles, such as the Teratocarcinoma Cell Line GH and breast cancer Cell Lines, recently melanoma Cells were described to express HERV-K proteins and particles. In order to study the expression of HERV-K, -H, -R and -W, in melanoma Cell Lines and biopsies primer sets were used. Antisera specific for HERV-K proteins were used for immunohistochemistry and sera from melanoma patients were investigated for HERV-K specific antibodies. Full length mRNAs of all HERVs were found in all human Cells. Spliced env and rec of HERV-K were detected in 39% of the melanoma biopsies and in 38% of the melanoma Cell Lines. Expression of HERV-K in situ was shown by immunohistochemistry. In addition, 16% of the patients sera tested showed antibodies against the HERV-K transmembrane envelope protein, but no antibodies against Np9 or Rec could be detected. A certain dedifferentiation of Cells as a consequence of tumour development is discussed. Therefore the expression of HERV-K in undifferentiated embryonic stem Cells was investigated. The investigated stem Cells showed expression of HERV-K full length, env, rec and np9 mRNA. Although the expression decreased with differentiation to neuronal precursor Cells. Even though HERV-K mRNA and proteins were expressed in a high percentage of melanomas their function in tumour development is still unclear. As well as the meaning of the HERV-K expression in embryonic stem Cells, particularly for a tumourigenic potential
-
Expression und biologische Funktion von humanen endogenen Retroviren (HERVs)
Humboldt-Universität zu Berlin Mathematisch-Naturwissenschaftliche Fakultät I, 2006Co-Authors: Büscher KristinaAbstract:Daten des humanen Genomprojektes zeigen, dass ca. 8% des gesamten humanen Genoms aus retroviralen Sequenzen besteht. Der überwiegende Teil dieser Proviren ist aufgrund verschiedener Mutationen defekt. Im Gegensatz zu allen anderen HERV Proviren scheinen einige HERV-K Proviren intakt zu sein und besitzen offene Leserahmen für alle viralen Proteine. Die Familie des humanen endogenen Retrovirus K HML2 umfasst ca. 30 eng verwandte Proviren. Zusätzlich zu den Strukturproteinen Gag und Env und der Reversen Transkriptase, exprimiert HERV-K zwei regulatorische Proteine, Rec und Np9. Beide sind im Nukleus lokalisiert und tumorigene Eigenschaften bzw. eine Expression in Assoziation mit Tumorgeweben wurde nachgewiesen. Neben Zelllinien, wie die Teratokarzinomzelllinie GH und einigen Brustkrebszelllinien, für die die Expression von HERV-K mRNA und die Produktion von Viruspartikeln bekannt ist, konnte die Expression von HERV-K Proteinen und Partikeln für Melanomzellen gezeigt werden. Volllängen mRNA von HERV-K war in allen untersuchten humanen Proben nachweisbar. Gespleißtes env und rec war in 39% der Gewebe und in 38% der Melanomzelllinien exprimiert. Zusätzlich werden HERV-H, -R und -W exprimiert. Von den auf spezifische Antikörper gegen HERV-K Proteine untersuchten Seren der Melanompatienten waren 16% positiv für das transmembrane Hüllprotein, jedoch reagierte kein Serum mit Re oder Np9. Da im Zuge der Entstehung von Tumoren immer auch eine Dedifferenzierung der entarteten Zellen diskutiert wird, wurde die Expression von HERVs in undifferenzierten, embryonalen Stammzellen bestimmt. In den untersuchten embryonalen Stammzellen lässt sich Volllängen mRNA, sowie gespleißte env, rec und np9 mRNA nachweisen. Während der Differenzierung zu neuronalen Vorläuferzellen sinkt die Expression jedoch wieder auf ein mit normalen Zellen vergleichbares Niveau. Obwohl gespleißte RNA und virale Proteine von HERV-K vor allem in Tumoren und Tumorzelllinien exprimiert werden, ist deren Funktion während der Tumorentstehung noch immer ungeklärt. Auch die Bedeutung der HERV-K Expression in humanen Stammzellen ist noch unklar, insbesondere in Hinblick auf eine mögliche Tumorigenität.In contrast to all other human endogenous retroviruses, proviruses of the human endogenous retrovirus family HERV-K have maintained open reading frames for all viral proteins. Although most proviruses are defective, structural proteins Gag and Env, the reverse transcriptase and two regulatory proteins, Rec and Np9, have been described. Rec resembles the Rev protein of HIV and tumourigenic potential was confirmed. Np9 as well is located in the nucleus and expression in association with tumour tissues was observed. Additionally to Cell Lines known to produce HERV-K virus particles, such as the Teratocarcinoma Cell Line GH and breast cancer Cell Lines, recently melanoma Cells were described to express HERV-K proteins and particles. In order to study the expression of HERV-K, -H, -R and -W, in melanoma Cell Lines and biopsies primer sets were used. Antisera specific for HERV-K proteins were used for immunohistochemistry and sera from melanoma patients were investigated for HERV-K specific antibodies. Full length mRNAs of all HERVs were found in all human Cells. Spliced env and rec of HERV-K were detected in 39% of the melanoma biopsies and in 38% of the melanoma Cell Lines. Expression of HERV-K in situ was shown by immunohistochemistry. In addition, 16% of the patients sera tested showed antibodies against the HERV-K transmembrane envelope protein, but no antibodies against Np9 or Rec could be detected. A certain dedifferentiation of Cells as a consequence of tumour development is discussed. Therefore the expression of HERV-K in undifferentiated embryonic stem Cells was investigated. The investigated stem Cells showed expression of HERV-K full length, env, rec and np9 mRNA. Although the expression decreased with differentiation to neuronal precursor Cells. Even though HERV-K mRNA and proteins were expressed in a high percentage of melanomas their function in tumour development is still unclear. As well as the meaning of the HERV-K expression in embryonic stem Cells, particularly for a tumourigenic potential
Evangelos Delivopoulos - One of the best experts on this subject based on the ideXlab platform.
-
First human hNT astrocytes patterned to single Cell resolution on parylene-C/Silicon dioxide substrates
2011 Annual International Conference of the IEEE Engineering in Medicine and Biology Society, 2011Co-Authors: Charles P Unsworth, Evangelos Delivopoulos, Euan S. Graham, Alan F MurrayAbstract:In our previous work we developed a successful protocol to pattern the human hNT neuron (derived from the human Teratocarcinoma Cell Line (hNT)) on parylene-C/SiO2 substrates. This communication, reports how we have successfully managed to pattern the supportive Cell to the neuron, the hNT astrocyte, on such substrates. Here we disseminate the nanofabrication, Cell differentiation and Cell culturing protocols necessary to successfully pattern the first human hNT astrocytes to single Cell resolution on parylene-C/SiO2 substrates. This is performed for varying parylene strip widths providing exCellent contrast to the SiO2 substrate and elegant single Cell isolation at 10μm strip widths. The breakthrough in patterning human Cells on a silicon chip has widespread implications and is valuable as a platform technology as it enables a detailed study of the human brain at the Cellular and network level.
-
first human hnt neurons patterned on parylene c silicon dioxide substrates combining an accessible Cell Line and robust patterning technology for the study of the pathological adult human brain
Journal of Neuroscience Methods, 2010Co-Authors: Charles P Unsworth, E Graham, Evangelos Delivopoulos, Mike Dragunow, Alan F MurrayAbstract:In this communication, we describe a new method which has enabled the first patterning of human neurons (derived from the human Teratocarcinoma Cell Line (hNT)) on parylene-C/silicon dioxide substrates. We reveal the details of the nanofabrication processes, Cell differentiation and culturing protocols necessary to successfully pattern hNT neurons which are each key aspects of this new method. The benefits in patterning human neurons on silicon chip using an accessible Cell Line and robust patterning technology are of widespread value. Thus, using a combined technology such as this will facilitate the detailed study of the pathological human brain at both the single Cell and network level.