Dataset Information

HiPSC-CM Monolayer Maturation State Determines Drug Responsiveness in High Throughput Pro-Arrhythmia Screen.

ABSTRACT: Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) offer a novel in vitro platform for pre-clinical cardiotoxicity and pro-arrhythmia screening of drugs in development. To date hiPSC-CMs used for cardiotoxicity testing display an immature, fetal-like cardiomyocyte structural and electrophysiological phenotype which has called into question the applicability of hiPSC-CM findings to the adult heart. The aim of the current work was to determine the effect of cardiomyocyte maturation state on hiPSC-CM drug responsiveness. To this end, here we developed a high content pro-arrhythmia screening platform consisting of either fetal-like or mature hiPSC-CM monolayers. Compounds tested in the screen were selected based on the pro-arrhythmia risk classification (Low risk, Intermediate risk, or High risk) established recently by the FDA and major stakeholders in the Drug Discovery field for the validation of the Comprehensive In vitro Pro-Arrhythmia Assay (CiPA). Here we show that maturation state of hiPSC-CMs determines the absolute pro-arrhythmia risk score calculated for these compounds. Thus, the maturation state of hiPSC-CMs should be considered prior to pro-arrhythmia and cardiotoxicity screening in drug discovery programs.

SUBMITTER: da Rocha AM

PROVIDER: S-EPMC5653750 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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hiPSC-CM Monolayer Maturation State Determines Drug Responsiveness in High Throughput Pro-Arrhythmia Screen.

da Rocha André Monteiro AM Campbell Katherine K Mironov Sergey S Jiang Jiang J Mundada Lakshmi L Guerrero-Serna Guadalupe G Jalife José J Herron Todd J TJ

Scientific reports 20171023 1

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) offer a novel in vitro platform for pre-clinical cardiotoxicity and pro-arrhythmia screening of drugs in development. To date hiPSC-CMs used for cardiotoxicity testing display an immature, fetal-like cardiomyocyte structural and electrophysiological phenotype which has called into question the applicability of hiPSC-CM findings to the adult heart. The aim of the current work was to determine the effect of cardiomyocyte matura ...[more]

PMID: 29061979

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Project description:AimsHuman-induced pluripotent stem cell-cardiomyocytes (hiPSC-CMs) are widely used to study arrhythmia-associated mutations in ion channels. Among these, the cardiac sodium channel SCN5A undergoes foetal-to-adult isoform switching around birth. Conventional hiPSC-CM cultures, which are phenotypically foetal, have thus far been unable to capture mutations in adult gene isoforms. Here, we investigated whether tri-cellular cross-talk in a three-dimensional (3D) cardiac microtissue (MT) promoted post-natal SCN5A maturation in hiPSC-CMs.Methods and resultsWe derived patient hiPSC-CMs carrying compound mutations in the adult SCN5A exon 6B and exon 4. Electrophysiological properties of patient hiPSC-CMs in monolayer were not altered by the exon 6B mutation compared with isogenic controls since it is not expressed; further, CRISPR/Cas9-mediated excision of the foetal exon 6A did not promote adult SCN5A expression. However, when hiPSC-CMs were matured in 3D cardiac MTs, SCN5A underwent isoform switch and the functional consequences of the mutation located in exon 6B were revealed. Up-regulation of the splicing factor muscleblind-like protein 1 (MBNL1) drove SCN5A post-natal maturation in microtissues since its overexpression in hiPSC-CMs was sufficient to promote exon 6B inclusion, whilst knocking-out MBNL1 failed to foster isoform switch.ConclusionsOur study shows that (i) the tri-cellular cardiac microtissues promote post-natal SCN5A isoform switch in hiPSC-CMs, (ii) adult splicing of SCN5A is driven by MBNL1 in these tissues, and (iii) this model can be used for examining post-natal cardiac arrhythmias due to mutations in the exon 6B.Translational perspectiveThe cardiac sodium channel is essential for conducting the electrical impulse in the heart. Postnatal alternative splicing regulation causes mutual exclusive inclusion of fetal or adult exons of the corresponding gene, SCN5A. Typically, immature hiPSCCMs fall short in studying the effect of mutations located in the adult exon. We describe here that an innovative tri-cellular three-dimensional cardiac microtissue culture promotes hiPSC-CMs maturation through upregulation of MBNL1, thus revealing the effect of a pathogenic genetic variant located in the SCN5A adult exon. These results help advancing the use of hiPSC-CMs in studying adult heart disease and for developing personalized medicine applications.

Project description:Macroautophagy/autophagy cytoplasmic quality control pathways are required during neural development and are critical for the maintenance of functional neuronal populations in the adult brain. Robust evidence now exists that declining neuronal autophagy pathways contribute to human neurodegenerative diseases, including Parkinson disease (PD). Reliable and relevant human neuronal model systems are therefore needed to understand the biology of disease-vulnerable neural populations, to decipher the underlying causes of neurodegenerative disease, and to develop assays to test therapeutic interventions in vitro. Human induced pluripotent stem cell (hiPSC) neural model systems can meet this demand: they provide a renewable source of material for differentiation into regional neuronal sub-types for functional assays; they can be expanded to provide a platform for screening, and they can potentially be optimized for transplantation/neurorestorative therapy. So far, however, hiPSC differentiation protocols for the generation of ventral midbrain dopaminergic neurons (mDANs) - the predominant neuronal sub-type afflicted in PD - have been somewhat restricted by poor efficiency and/or suitability for functional and/or imaging-based in vitro assays. Here, we describe a reliable, monolayer differentiation protocol for the rapid and reproducible production of high numbers of mDANs from hiPSC in a format that is amenable for autophagy/mitophagy research. We characterize these cells with respect to neuronal differentiation and macroautophagy capability and describe qualitative and quantitative assays for the study of autophagy and mitophagy in these important cells.Abbreviations: AA: ascorbic acid; ATG: autophagy-related; BDNF: brain derived neurotrophic factor; CCCP: carbonyl cyanide m-chlorophenylhydrazone; dbcAMP: dibutyryl cAMP; DAN: dopaminergic neuron; DAPI: 4',6-diamidino-2-phenylindole; DAPT: N-[N-(3,5-difluorophenacetyl)-L-alanyl]-sphenylglycine; DLG4/PSD95: discs large MAGUK scaffold protein 4; DMEM: Dulbecco's modified eagle's medium; EB: embryoid body; ECAR: extracellular acidification rate; EGF: epidermal growth factor; FACS: fluorescence-activated cell sorting; FCCP: arbonyl cyanide p-triflouromethoxyphenylhydrazone; FGF: fibroblast growth factor; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GDNF: glia cell derived neurotrophic factor; hiPSC: human induced pluripotent stem cell; LAMP2A: lysosomal associated membrane protein 2A; LT-R: LysoTracker Red; MAP1LC3: microtubule associated protein 1 light chain 3; mDAN: midbrain dopaminergic neuron; MEF: mouse embryonic fibroblast; MT-GR: MitoTracker Green; MT-R: MitoTracker Red; NAS2: normal SNCA2; NEM: neuroprogenitor expansion media; NR4A2/NURR1: nuclear receptor subfamily group A member 2; OA: oligomycin and antimycin A; OCR: oxygen consumption rate; PD: Parkinson disease; SHH: sonic hedgehog signaling molecule; SNCA/α-synuclein: synuclein alpha; TH: tyrosine hydroxylase; VTN: vitronectin.

Dataset Information

HiPSC-CM Monolayer Maturation State Determines Drug Responsiveness in High Throughput Pro-Arrhythmia Screen.

Publications

hiPSC-CM Monolayer Maturation State Determines Drug Responsiveness in High Throughput Pro-Arrhythmia Screen.

Similar Datasets

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