scholarly journals hiPSC-Derived Cardiomyocyte Model of LQT2 Syndrome Derived from Asymptomatic and Symptomatic Mutation Carriers Reproduces Clinical Differences in Aggregates but Not in Single Cells

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1153 ◽  
Author(s):  
Disheet Shah ◽  
Chandra Prajapati ◽  
Kirsi Penttinen ◽  
Reeja Maria Cherian ◽  
Jussi T. Koivumäki ◽  
...  
Keyword(s):  
Single Cells ◽  
Induced Pluripotent Stem Cell ◽  
Genetic Mutation ◽  
Channel Blockers ◽  
Aggregate Level ◽  
Gene Encoding ◽  
Clinical Phenotypes ◽  
Mutation Carriers ◽  
Induced Pluripotent

Mutations in the HERG gene encoding the potassium ion channel HERG, represent one of the most frequent causes of long QT syndrome type-2 (LQT2). The same genetic mutation frequently presents different clinical phenotypes in the family. Our study aimed to model LQT2 and study functional differences between the mutation carriers of variable clinical phenotypes. We derived human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) from asymptomatic and symptomatic HERG mutation carriers from the same family. When comparing asymptomatic and symptomatic single LQT2 hiPSC-CMs, results from allelic imbalance, potassium current density, and arrhythmicity on adrenaline exposure were similar, but a difference in Ca2+ transients was observed. The major differences were, however, observed at aggregate level with increased susceptibility to arrhythmias on exposure to adrenaline or potassium channel blockers on CM aggregates derived from the symptomatic individual. The effect of this mutation was modeled in-silico which indicated the reactivation of an inward calcium current as one of the main causes of arrhythmia. Our in-vitro hiPSC-CM model recapitulated major phenotype characteristics observed in LQT2 mutation carriers and strong phenotype differences between LQT2 asymptomatic vs. symptomatic were revealed at CM-aggregate level.

scholarly journals Deep learning detects cardiotoxicity in a high-content screen with induced pluripotent stem cell-derived cardiomyocytes

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Francis Grafton ◽  
Jaclyn Ho ◽  
Sara Ranjbarvaziri ◽  
Farshad Farshidfar ◽  
Anastasiia Budan ◽  
...  
Keyword(s):  
Stem Cell ◽  
Deep Learning ◽  
Pluripotent Stem Cell ◽  
Kinase Inhibitors ◽  
Early Stage ◽  
Induced Pluripotent Stem Cell ◽  
Channel Blockers ◽  
Drug Induced ◽  
Induced Pluripotent

Drug-induced cardiotoxicity and hepatotoxicity are major causes of drug attrition. To decrease late-stage drug attrition, pharmaceutical and biotechnology industries need to establish biologically relevant models that use phenotypic screening to detect drug-induced toxicity in vitro. In this study, we sought to rapidly detect patterns of cardiotoxicity using high-content image analysis with deep learning and induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). We screened a library of 1280 bioactive compounds and identified those with potential cardiotoxic liabilities in iPSC-CMs using a single-parameter score based on deep learning. Compounds demonstrating cardiotoxicity in iPSC-CMs included DNA intercalators, ion channel blockers, epidermal growth factor receptor, cyclin-dependent kinase, and multi-kinase inhibitors. We also screened a diverse library of molecules with unknown targets and identified chemical frameworks that show cardiotoxic signal in iPSC-CMs. By using this screening approach during target discovery and lead optimization, we can de-risk early-stage drug discovery. We show that the broad applicability of combining deep learning with iPSC technology is an effective way to interrogate cellular phenotypes and identify drugs that may protect against diseased phenotypes and deleterious mutations.

scholarly journals Presynaptic dysfunction in CASK-related neurodevelopmental disorders

2019 ◽  
Author(s):  
Martin Becker ◽  
Francesca Mastropasqua ◽  
Jan Philipp Reising ◽  
Simon Maier ◽  
Mai-Lan Ho ◽  
...  
Keyword(s):  
Induced Pluripotent Stem Cell ◽  
Resonance Spectroscopy ◽  
Limited Information ◽  
Mutation Carriers ◽  
Gene Sets ◽  
Human Neurons ◽  
Induced Pluripotent ◽  
Human Neuronal Cells

SummaryCASK-related disorders are a genetically defined group of neurodevelopmental syndromes. There is limited information about the effects of CASK mutations in human neurons. Therefore, we sought to delineate CASK mutation consequences and neuronal level effects using induced pluripotent stem cell-derived neurons from two mutation carriers; one male diagnosed with ASD and a female with MICPCH. We show a reduction of the CASK protein in maturing neurons from the mutation carriers, which leads to significant downregulation of gene sets involved in presynaptic development and CASK protein interactors. Furthermore, CASK-deficient neurons showed decreased inhibitory presynapse size as indicated by VGAT staining, which may alter the excitatory-inhibitory (E/I) balance in developing neural circuitries. Using in vivo magnetic resonance spectroscopy quantification of GABA in the male mutation carrier, we further highlight the possibility to validate in vitro cellular data in brain. Our data shows that future pharmacological and clinical studies on targeting presynapses and E/I imbalance could lead to specific treatments for CASK-related disorders.HighlightsModelling of CASK-related disorders using iPSC-derived human neuronal cellsCASK mutations cause dysregulation of its protein interactor partnersReduced CASK levels primarily affect inhibitory presynapse developmentIn vitro GABAergic phenotype predicts in vivo neurotransmitter levels

scholarly journals Determination of Appropriate Stage of Human-Induced Pluripotent Stem Cell–Derived Cardiomyocytes for Drug Screening and Pharmacological Evaluation In Vitro

2012 ◽  
Vol 17 (9) ◽  
pp. 1192-1203 ◽  
Author(s):  
Tadahiro Shinozawa ◽  
Kenichi Imahashi ◽  
Hiroshi Sawada ◽  
Hatsue Furukawa ◽  
Kenji Takami
Keyword(s):  
Stem Cell ◽  
Sarcoplasmic Reticulum ◽  
Pluripotent Stem Cell ◽  
Microelectrode Array ◽  
Contractile Function ◽  
Induced Pluripotent Stem Cell ◽  
Channel Blockers ◽  
Electrophysiological Properties ◽  
Induced Pluripotent

Human-induced pluripotent stem cell–derived cardiomyocytes (hiPS-CMs) at different stages (approximate days 30, 60, and 90) were used to determine the appropriate stage for functional and morphological assessment of drug effects in vitro. The hiPS-CMs had spontaneous beating activity, and β-adrenergic function was comparable in all stages of differentiation. Microelectrode array analyses using ion channel blockers indicated that the electrophysiological properties of these ion channels were comparable at all differentiation stages. Ultrastructural analysis using electron microscopy showed that myofibrillar structures at days 60 and 90 were similarly distributed and more mature than that at day 30. Analysis of motion vectors in contracting cells showed that the velocity of contraction was the highest at day 90 and was the most mature among the three stages. Gene expression analysis demonstrated that expression of some genes related to myofilament and sarcoplasmic reticulum increased with maturation of morphological and contractile properties. In conclusion, day 30 cardiomyocytes are useful for basic screening such as the assessment of electrophysiological properties, and days 60 and 90 are the appropriate differentiation stage for morphological assays. For the assay of contractile function associated with subcellular components such as sarcoplasmic reticulum, day 90 cardiomyocytes are the most suitable.

Wiskott-Aldrich syndrome iPS cells produce megakaryocytes with defects in cytoskeletal rearrangement and proplatelet formation

2015 ◽  
Vol 113 (04) ◽  
pp. 792-805 ◽  
Author(s):  
Praewphan Ingrungruanglert ◽  
Pramuk Amarinthnukrowh ◽  
Ruttachuk Rungsiwiwut ◽  
Supang Maneesri-le Grand ◽  
Darintr Sosothikul ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Lentiviral Vector ◽  
Induced Pluripotent Stem Cell ◽  
Gene Encoding ◽  
Wiskott Aldrich Syndrome ◽  
Cytoskeletal Rearrangement ◽  
Irregular Shapes ◽  
Proplatelet Formation ◽  
Induced Pluripotent

SummaryWiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterised by microthrombocytopenia, complex immunodeficiency, autoimmunity, and haematologic malignancies. It is caused by mutations in the gene encoding WAS protein (WASP), a regulator of actin cytoskeleton and chromatin structure in various blood cell lineages. The molecular mechanisms underlying microthrombocytopenia caused by WASP mutations remain elusive. Murine models of WASP deficiency exhibited only mild thrombocytopenia with normal-sized platelets. Here we report on the successful generation of induced pluripotent stem cell (iPSC) lines from two patients with different mutations in WASP (c.1507T>A and c.55C>T). When differentiated into early CD34+ haematopoietic and megakaryocyte progenitors, the WAS-iPSC lines were indistinguishable from the wild-type iPSCs. However, all WAS-iPSC lines exhibited defects in platelet production in vitro. WAS-iPSCs produced platelets with more irregular shapes and smaller sizes. Immunofluorescence and electron micrograph showed defects in cytoskeletal rearrangement, F-actin distribution, and proplatelet formation. Proplatelet defects were more pronounced when using culture systems with stromal feeders comparing to feeder-free culture condition. Overexpression of WASP in the WAS-iPSCs using a lentiviral vector improved proplatelet structures and increased the platelet size. Our findings substantiate the use of iPSC technology to elucidate the disease mechanisms of WAS in thrombopoiesis.

Use of 3D culture systems to generate human induced pluripotent stem cell-derived [beta]-cells in vitro

2018 ◽  
Author(s):  
Fantuzzi Federica ◽  
Toivonen Sanna ◽  
Schiavo Andrea Alex ◽  
Pachera Nathalie ◽  
Rajaei Bahareh ◽  
...  
Keyword(s):  
Stem Cell ◽  
Beta Cells ◽  
Pluripotent Stem Cell ◽  
3D Culture ◽  
Induced Pluripotent Stem Cell ◽  
Culture Systems ◽  
Induced Pluripotent

scholarly journals Bestrophinopathies: perspectives on clinical disease, Bestrophin-1 function and developing therapies

2021 ◽  
Vol 13 ◽  
pp. 251584142199719
Author(s):  
Simranjeet Singh Grewal ◽  
Joseph J. Smith ◽  
Amanda-Jayne F. Carr
Keyword(s):  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Induced Pluripotent Stem Cell ◽  
Underlying Disease ◽  
Incomplete Penetrance ◽  
Inherited Retinal Dystrophies ◽  
Retinal Dystrophies ◽  
High Acuity ◽  
Induced Pluripotent

Bestrophinopathies are a group of clinically distinct inherited retinal dystrophies that typically affect the macular region, an area synonymous with central high acuity vision. This spectrum of disorders is caused by mutations in bestrophin1 ( BEST1), a protein thought to act as a Ca2+-activated Cl- channel in the retinal pigment epithelium (RPE) of the eye. Although bestrophinopathies are rare, over 250 individual pathological mutations have been identified in the BEST1 gene, with many reported to have various clinical expressivity and incomplete penetrance. With no current clinical treatments available for patients with bestrophinopathies, understanding the role of BEST1 in cells and the pathological pathways underlying disease has become a priority. Induced pluripotent stem cell (iPSC) technology is helping to uncover disease mechanisms and develop treatments for RPE diseases, like bestrophinopathies. Here, we provide a comprehensive review of the pathophysiology of bestrophinopathies and highlight how patient-derived iPSC-RPE are being used to test new genomic therapies in vitro.

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