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  1. Article ; Online: High-Throughput Screening Assay for Detecting Drug-Induced Changes in Synchronized Neuronal Oscillations and Potential Seizure Risk Based on Ca 2+ Fluorescence Measurements in Human Induced Pluripotent Stem Cell (hiPSC)-Derived Neuronal 2D and 3D Cultures

    Hua-Rong Lu / Manabu Seo / Mohamed Kreir / Tetsuya Tanaka / Rie Yamoto / Cristina Altrocchi / Karel van Ammel / Fetene Tekle / Ly Pham / Xiang Yao / Ard Teisman / David J. Gallacher

    Cells, Vol 12, Iss 958, p

    2023  Volume 958

    Abstract: Drug-induced seizure liability is a significant safety issue and the basis for attrition in drug development. Occurrence in late development results in increased costs, human risk, and delayed market availability of novel therapeutics. Therefore, there ... ...

    Abstract Drug-induced seizure liability is a significant safety issue and the basis for attrition in drug development. Occurrence in late development results in increased costs, human risk, and delayed market availability of novel therapeutics. Therefore, there is an urgent need for biologically relevant, in vitro high-throughput screening assays (HTS) to predict potential risks for drug-induced seizure early in drug discovery. We investigated drug-induced changes in neural Ca 2+ oscillations, using fluorescent dyes as a potential indicator of seizure risk, in hiPSC-derived neurons co-cultured with human primary astrocytes in both 2D and 3D forms. The dynamics of synchronized neuronal calcium oscillations were measured with an FDSS kinetics reader. Drug responses in synchronized Ca 2+ oscillations were recorded in both 2D and 3D hiPSC-derived neuron/primary astrocyte co-cultures using positive controls (4-aminopyridine and kainic acid) and negative control (acetaminophen). Subsequently, blinded tests were carried out for 25 drugs with known clinical seizure incidence. Positive predictive value (accuracy) based on significant changes in the peak number of Ca 2+ oscillations among 25 reference drugs was 91% in 2D vs. 45% in 3D hiPSC-neuron/primary astrocyte co-cultures. These data suggest that drugs that alter neuronal activity and may have potential risk for seizures can be identified with high accuracy using an HTS approach using the measurements of Ca 2+ oscillations in hiPSC-derived neurons co-cultured with primary astrocytes in 2D.
    Keywords hiPSC neurons ; 2D ; 3D ; high throughput screening HTS ; Ca 2+ neuronal oscillations ; neuronal active drugs ; Biology (General) ; QH301-705.5
    Subject code 333
    Language English
    Publishing date 2023-03-01T00:00:00Z
    Publisher MDPI AG
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  2. Article ; Online: Development of a fully human assay combining NGN2-inducible neurons co-cultured with iPSC-derived astrocytes amenable for electrophysiological studies

    Pei-Yu Shih / Mohamed Kreir / Devesh Kumar / Frederik Seibt / Francisco Pestana / Benjamin Schmid / Bjørn Holst / Christian Clausen / Rachel Steeg / Benjamin Fischer / Juan Pita-Almenar / Andreas Ebneth / Alfredo Cabrera-Socorro

    Stem Cell Research, Vol 54, Iss , Pp 102386- (2021)

    2021  

    Abstract: Neurogenin 2 encodes a neural-specific transcription factor (NGN2) able to drive neuronal fate on somatic and stem cells. NGN2 is expressed in neural progenitors within the developing central and peripheral nervous systems. Overexpression of NGN2 in ... ...

    Abstract Neurogenin 2 encodes a neural-specific transcription factor (NGN2) able to drive neuronal fate on somatic and stem cells. NGN2 is expressed in neural progenitors within the developing central and peripheral nervous systems. Overexpression of NGN2 in human induced pluripotent stem cells (hiPSCs) or human embryonic stem cells has been shown to efficiently trigger conversion to neurons. Here we describe two gene-edited hiPSC lines harbouring a doxycycline (DOX)-inducible cassette in the AAVS1 locus driving expression of NGN2 (BIONi010-C-13) or NGN2-T2A-GFP (BIONi010-C-15). By introducing NGN2-expressing cassette, we reduce variability associated with conventional over-expression methods such as viral transduction, making these lines amenable for scale-up production and screening processes. DOX-treated hiPSCs convert to neural phenotype within one week and display the expression of structural neuronal markers such as Beta-III tubulin and tau. We performed functional characterization of NGN2-neurons co-cultured with hiPSC-derived astrocytes in a “fully-humanized” set up. Passive properties of NGN2-neurons were indistinguishable from mouse primary cells while displaying variable activity in extracellular recordings performed in multi-electrode arrays (MEAs). We demonstrate that hiPSC-derived astrocytes and neurons can be co-cultured and display functional properties comparable to the gold standard used in electrophysiology. Both lines are globally available via EBiSC repository at https://cells.ebisc.org/.
    Keywords Biology (General) ; QH301-705.5
    Subject code 571
    Language English
    Publishing date 2021-07-01T00:00:00Z
    Publisher Elsevier
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  3. Article: Membrane assembly of the functional KcsA potassium channel in a vesicle-based eukaryotic cell-free translation system

    Dondapati, Srujan Kumar / Andrea Brüggemann / Doreen A Wüstenhagen / Mohamed Kreir / Niels Fertig / Robert B. Quast / Stefan Kubick

    Biosensors & bioelectronics. 2014 Sept. 15, v. 59

    2014  

    Abstract: The potassium channel KcsA was heterologously expressed in a eukaryotic cell-free system. Both, the expression yields and functional analysis of the protein were reported. Qualitative and quantitative analyses of KcsA expression were performed by using ... ...

    Abstract The potassium channel KcsA was heterologously expressed in a eukaryotic cell-free system. Both, the expression yields and functional analysis of the protein were reported. Qualitative and quantitative analyses of KcsA expression were performed by using 14C-labeled leucine as one of the amino acids supplemented in the cell-free reaction mixture. There was a time dependent increase in the protein yield as well as the intensity of the native tetramer band in insect cell derived microsomes. Electrophysiology measurements demonstrated the functional activity of the microsomes harboring KcsA showing single-channel currents with the typical biophysical characteristics of the ion channel. The channel behavior was asymmetric and showed positive rectification with larger currents towards positive voltages. KcsA channel currents were effectively blocked by potassium selective barium (Ba2+). This functional demonstration of an ion channel in eukaryotic cell-free system has a large potential for future applications including drug screening, diagnostic applications and functional assessment of complex membrane proteins like GPCRs by coupling them to ion channels in cell-free systems. Furthermore, membrane proteins can be expressed directly from linear DNA templates within 90min, eliminating the need for additional cloning steps, which makes this cell-free system fast and efficient.
    Keywords barium ; biosensors ; cell free system ; DNA ; drugs ; electric power ; electrophysiology ; insects ; leucine ; microsomes ; potassium ; potassium channels ; quantitative analysis ; screening
    Language English
    Dates of publication 2014-0915
    Size p. 174-183.
    Publishing place Elsevier B.V.
    Document type Article
    ZDB-ID 1011023-9
    ISSN 1873-4235 ; 0956-5663
    ISSN (online) 1873-4235
    ISSN 0956-5663
    DOI 10.1016/j.bios.2014.03.004
    Database NAL-Catalogue (AGRICOLA)

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