10 research outputs found

    Generation of human induced pluripotent stem cell line from a patient with a long QT syndrome type 2

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    We report here the generation of human iPS cell line UKKi009-A from dermal fibroblasts of a patient carrying heterozygous mutation c.3035-3045delTCCCTCGATGC, p.Leu1012Pro (fs*55) in KCNH2 gene leading to long QT syndrome type 2 (LQT2). We used the Sleeping Beauty transposon-based plasmids expressing OSKM along with microRNAs 307/367 to reprogram the fibroblasts. The iPS cells possess pluripotent stem cell characteristics and differentiate to cell lineages of all three germ layers. This cell line can serve as a source for in vitro modeling of LQT2. This cell line is distributed by the European Collection of Authenticated Cell Cultures (ECACC)

    Fabrication of High-Entropy Alloys Using a Combination of Detonation Spraying and Spark Plasma Sintering: A Case Study Using the Al-Fe-Co-Ni-Cu System

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    The use of pre-alloyed powders as high-entropy alloy (HEA) coating precursors ensures a predetermined (unaltered) elemental composition of the coating with regard to the feedstock powder. At the same time, it is interesting to tackle a more challenging task: to form alloy coatings from powder blends (not previously alloyed). The powder-blend-based route of coating formation eliminates the need to use atomization or ball milling equipment for powder preparation and allows for the introduction of additives into the material in a flexible manner. In this work, for the first time, a HEA was obtained using detonation spraying (DS) followed by spark plasma sintering (SPS). A powder mixture with a nominal composition of 10Al-22.5Fe-22.5Co-22.5Ni-22.5Cu (at.%) was detonation-sprayed to form a multicomponent metallic coating on a steel substrate. The elemental composition of the deposited layer was (9 ± 1)Al-(10 ± 1)Fe-(20 ± 1)Co-(34 ± 1)Ni-(27 ± 1)Cu (at.%), which is different from that of the feedstock powder because of the differences in the deposition efficiencies of the metals during DS. Despite the compositional deviations, the deposited layer was still suitable as a precursor for a HEA with a configurational entropy of ~1.5R, where R is the universal gas constant. The subsequent SPS treatment of the substrate/coating assembly was carried out at 800–1000 °C at a uniaxial pressure of 40 MPa. The SPS treatment of the deposited layer at 1000 °C for 20 min was sufficient to produce an alloy with a single-phase face-centered cubic structure and a porosity of 0.3). The hardness of the coatings measured in two perpendicular directions did not differ significantly. The features of the DS–SPS route of the formation of HEA coatings and its potential applications are discussed

    Ascorbic Acid-Induced Cardiac Differentiation of Murine Pluripotent Stem Cells: Transcriptional Profiling and Effect of a Small Molecule Synergist of Wnt/β-Catenin Signaling Pathway

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    Background: Reproducible and efficient differentiation of pluripotent stem cells (PSCs) to cardiomyocytes (CMs) is essential for their use in regenerative medicine, drug testing and disease modeling. The aim of this study was to evaluate the effect of some previously reported cardiogenic substances on cardiac differentiation of mouse PSCs. Methods: Differentiation was performed by embryoid body (EB)-based method using three different murine PSC lines. The differentiation efficiency was monitored by RT-qPCR, immunocytochemistry and flow cytometry, and the effect mechanistically evaluated by transcriptome analysis of treated EBs. Results: Among the five tested compounds (ascorbic acid, dorsomorphin, cyclic adenosine 3',5'-monophosphate, cardiogenol C, cyclosporin A) only ascorbic acid (AA) exerted a strong and reproducible cardiogenic effect in CGR8 cells which was less consistent in other two PSC lines. AA induced only minor changes in transcriptome of CGR8 cells after administration during the initial two days of differentiation. Cardiospecific genes and transcripts involved in angiogenesis, erythropoiesis and hematopoiesis were up-regulated on day 5 but not on days 2 or 3 of differentiation. The cardiac differentiation efficiency was improved when QS11, a small-molecule synergist of Wnt/β-catenin signaling pathway, was added to cultures after AA-treatment. Conclusion: This study demonstrates that only minor transcriptional changes are sufficient for enhancement of cardiogenesis of murine PSCs by AA and that AA and QS11 exhibit synergistic effects and enhance the efficiency of CM differentiation of murine PSCs

    Electrophysiological Properties of Tetraploid Cardiomyocytes Derived from Murine Pluripotent Stem Cells Generated by Fusion of Adult Somatic Cells with Embryonic Stem Cells

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    Most cardiomyocytes (CMs) in the adult mammalian heart are either binucleated or contain a single polyploid nucleus. Recent studies have shown that polyploidy in CMs plays an important role as an adaptive response to physiological demands and environmental stress and correlates with poor cardiac regenerative ability after injury. However, knowledge about the functional properties of polyploid CMs is limited. In this study, we generated tetraploid pluripotent stem cells (PSCs) by fusion of murine embryonic stem cells (ESCs) and somatic cells isolated from bone marrow or spleen and performed a comparative analysis of the electrophysiological properties of tetraploid fusion-derived PSCs and diploid ESC-derived CMs. Fusion-derived PSCs exhibited characteristics of genuine ESCs and contained a near-tetraploid genome. Ploidy features and marker expression were also retained during the differentiation of fusion-derived cells. Fusion-derived PSCs gave rise to CMs, which were similar to their diploid ESC counterparts in terms of their expression of typical cardiospecific markers, sarcomeric organization, action potential parameters, response to pharmacologic stimulation with various drugs, and expression of functional ion channels. These results suggest that the state of ploidy does not significantly affect the structural and electrophysiological properties of murine PSC-derived CMs. These results extend our knowledge of the functional properties of polyploid CMs and contribute to a better understanding of their biological role in the adult heart

    Interferon-gamma signaling synergizes with LRRK2 in neurons and microglia derived from human induced pluripotent stem cells

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    Parkinson's disease-associated kinase LRRK2 has been linked to IFN type II (IFN-γ) response in infections and to dopaminergic neuronal loss. However, whether and how LRRK2 synergizes with IFN-γ remains unclear. In this study, we employed dopaminergic neurons and microglia differentiated from patient-derived induced pluripotent stem cells carrying LRRK2 G2019S, the most common Parkinson's disease-associated mutation. We show that IFN-γ enhances the LRRK2 G2019S-dependent negative regulation of AKT phosphorylation and NFAT activation, thereby increasing neuronal vulnerability to immune challenge. Mechanistically, LRRK2 G2019S suppresses NFAT translocation via calcium signaling and possibly through microtubule reorganization. In microglia, LRRK2 modulates cytokine production and the glycolytic switch in response to IFN-γ in an NFAT-independent manner. Activated LRRK2 G2019S microglia cause neurite shortening, indicating that LRRK2-driven immunological changes can be neurotoxic. We propose that synergistic LRRK2/IFN-γ activation serves as a potential link between inflammation and neurodegeneration in Parkinson's disease.status: publishe

    Interferon-gamma signaling synergizes with LRRK2 in neurons and microglia derived from human induced pluripotent stem cells

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    IFN-γ signalling is linked to regional neuronal vulnerability in Parkinson’s disease. The authors show that a PD-associated pathogenic LRRK2 missense mutation increases neuronal susceptibility to immune challenges via negative regulation of AKT phosphorylation and NFAT activation in human iPSC-derived neurons and microglia
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