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  • Human induced pluripotent stem cell line with genetically encoded fluorescent voltage indicator generated via CRISPR for action potential assessment post-cardiogenesis
Title: Human induced pluripotent stem cell line with genetically encoded fluorescent voltage indicator generated via CRISPR for action potential assessment post-cardiogenesis
Abstract

Genetically encoded fluorescent voltage indicators, such as ArcLight, have been used to report action potentials (APs) in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). However, the ArcLight expression, in all cases, relied on a high number of lentiviral vector-mediated random genome integrations (8-12 copy/cell), raising concerns such as gene disruption and alteration of global and local gene expression, as well as loss or silencing of reporter genes after differentiation. Here, we report the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease technique to develop a hiPSC line stably expressing ArcLight from the AAVS1 safe harbor locus. The hiPSC line retained proliferative ability with a growth rate similar to its parental strain. Optical recording with conventional epifluorescence microscopy allowed the detection of APs as early as 21 days postdifferentiation, and could be repeatedly monitored for at least 5 months. Moreover, quantification and analysis of the APs of ArcLight-CMs identified two distinctive subtypes: a group with high frequency of spontaneous APs of small amplitudes that were pacemaker-like CMs and a group with low frequency of automaticity and large amplitudes that resembled the working CMs. Compared with FluoVolt voltage-sensitive dye, although dimmer, the ArcLight reporter exhibited better optical performance in terms of phototoxicity and photostability with comparable sensitivities and signal-to-noise ratios. The hiPSC line with targeted ArcLight engineering design represents a useful tool for studying cardiac development or hiPSC-derived cardiac disease models and drug testing.

</sec> </span> <a href='#' class='show open-abstract' style='margin-left:10px;'>more »</a> <a href='#' class='hide close-abstract' style='margin-left:10px;'>« less</a> <div style="clear:both;margin-bottom:20px;"></div> <dl class="dl-horizontal small"> <dt>NSF-PAR ID:</dt> <dd>10363338</dd> </dl> <dl class="dl-horizontal small"> <dt>Author(s) / Creator(s):</dt> <dd> <a target="_blank" rel="noopener noreferrer" href="https://par.nsf.gov/search/author:"Sun, Yao-Hui""><span class="author" itemprop="author">Sun, Yao-Hui</span> <sup class="text-muted"></sup></a><span class="sep">; </span><a target="_blank" rel="noopener noreferrer" href="https://par.nsf.gov/search/author:"Kao, Hillary K. J.""><span class="author" itemprop="author">Kao, Hillary K. J.</span> <sup class="text-muted"></sup></a><span class="sep">; </span><a target="_blank" rel="noopener noreferrer" href="https://par.nsf.gov/search/author:"Chang, Che-Wei""><span class="author" itemprop="author">Chang, Che-Wei</span> <sup class="text-muted"></sup></a><span class="sep">; </span><a target="_blank" rel="noopener noreferrer" href="https://par.nsf.gov/search/author:"Merleev, Alexander""><span class="author" itemprop="author">Merleev, Alexander</span> <sup class="text-muted"></sup></a><span class="sep">; </span><a target="_blank" rel="noopener noreferrer" href="https://par.nsf.gov/search/author:"Overton, James L.""><span class="author" itemprop="author">Overton, James L.</span> <sup class="text-muted"></sup></a><span class="sep">; </span><a target="_blank" rel="noopener noreferrer" href="https://par.nsf.gov/search/author:"Pretto, Dalyir""><span class="author" itemprop="author">Pretto, Dalyir</span> <sup class="text-muted"></sup></a><span class="sep">; </span><a target="_blank" rel="noopener noreferrer" href="https://par.nsf.gov/search/author:"Yechikov, Sergey""><span class="author" itemprop="author">Yechikov, Sergey</span> <sup class="text-muted"></sup></a><span class="sep">; </span><a target="_blank" rel="noopener noreferrer" href="https://par.nsf.gov/search/author:"Maverakis, Emanual""><span class="author" itemprop="author">Maverakis, Emanual</span> <sup class="text-muted"></sup></a><span class="sep">; </span><a target="_blank" rel="noopener noreferrer" href="https://par.nsf.gov/search/author:"Chiamvimonvat, Nipavan""><span class="author" itemprop="author">Chiamvimonvat, Nipavan</span> <sup class="text-muted"></sup></a><span class="sep">; </span><a target="_blank" rel="noopener noreferrer" href="https://par.nsf.gov/search/author:"Chan, James W.""><span class="author" itemprop="author">Chan, James W.</span> <sup class="text-muted"></sup></a><span class="sep">; </span><a target="_blank" rel="noopener noreferrer" href="https://par.nsf.gov/search/author:"Lieu, Deborah K.""><span class="author" itemprop="author">Lieu, Deborah K.</span> <sup class="text-muted"></sup></a></dd> </dl> <dl class="dl-horizontal small"> <dt>Publisher / Repository:</dt> <dd itemprop="publisher">Oxford University Press</dd> </dl> <dl class="dl-horizontal small"> <dt>Date Published:</dt> <dd> <time itemprop="datePublished" datetime="2019-09-30">2019-09-30</time> </dd> </dl> <dl class="dl-horizontal small"> <dt>Journal Name:</dt> <dd>Stem Cells</dd> </dl> <dl class="dl-horizontal small"> <dt>Volume:</dt> <dd>38</dd> </dl> <dl class="dl-horizontal small"> <dt>Issue:</dt> <dd>1</dd> </dl> <dl class="dl-horizontal small"> <dt>ISSN:</dt> <dd>1066-5099</dd> </dl> <dl class="dl-horizontal small"> <dt>Page Range / eLocation ID:</dt> <dd>p. 90-101</dd> </dl> <dl class="dl-horizontal small"> <dt>Format(s):</dt> <dd>Medium: X</dd> </dl> <dl class="dl-horizontal small"> <dt>Sponsoring Org:</dt> <dd itemprop="sourceOrganization">National Science Foundation</dd> </dl> <div class="clearfix"></div> </div> </div> <div id="citation-addl" class="hidden-print"> <h5 id='mlt-header'>More Like this</h5> <ol class="item-list documents" id="citation-mlt" style="min-height: 80px;"> <li> <div class="article item document" itemscope itemtype="http://schema.org/TechArticle"> <div class="item-info"> <div class="title"> <a href="https://par.nsf.gov/biblio/10224739-syncytium-cell-growth-increases-kir2-contribution-human-ipsc-cardiomyocytes" itemprop="url"> <span class='span-link' itemprop="name">Syncytium cell growth increases Kir2.1 contribution in human iPSC-cardiomyocytes</span> </a> </div> <div> <strong> <a class="misc external-link" href="https://doi.org/10.1152/ajpheart.00148.2020" target="_blank" title="Link to document DOI">https://doi.org/10.1152/ajpheart.00148.2020  <span class="fas fa-external-link-alt"></span></a> </strong> </div> <div class="metadata"> <span class="authors"> <span class="author" itemprop="author">Li, Weizhen</span> <span class="sep">; </span><span class="author" itemprop="author">Han, Julie L.</span> <span class="sep">; </span><span class="author" itemprop="author">Entcheva, Emilia</span> </span> <span class="year">( <time itemprop="datePublished" datetime="2020-11-01">November 2020</time> , American Journal of Physiology-Heart and Circulatory Physiology) </span> </div> <span class="editors"> <span class="editor" itemprop="editor">null</span> (Ed.) </span> <div style="cursor: pointer;-webkit-line-clamp: 5;" class="abstract" itemprop="description"> Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) enable cardiotoxicity testing and personalized medicine. However, their maturity is of concern, including relatively depolarized resting membrane potential and more spontaneous activity compared with adult cardiomyocytes, implicating low or lacking inward rectifier potassium current ( I k1 ). Here, protein quantification confirms Kir2.1 expression in hiPSC-CM syncytia, albeit several times lower than in adult heart tissue. We find that hiPSC-CM culture density influences Kir2.1 expression at the mRNA level (potassium inwardly rectifying channel subfamily J member 2) and at the protein level and its associated electrophysiology phenotype. Namely, all-optical cardiac electrophysiology and pharmacological treatments reveal reduction of spontaneous and irregular activity and increase in action potential upstroke in denser cultures. Blocking I k1 -like currents with BaCl 2 increased spontaneous frequency and blunted action potential upstrokes during pacing in a dose-dependent manner only in the highest-density cultures, in line with I k1 ’s role in regulating the resting membrane potential. Our results emphasize the importance of syncytial growth of hiPSC-CMs for more physiologically relevant phenotype and the power of all-optical electrophysiology to study cardiomyocytes in their multicellular setting. NEW & NOTEWORTHY We identify cell culture density and cell-cell contact as an important factor in determining the expression of a key ion channel at the transcriptional and the protein levels, KCNJ2/Kir2.1, and its contribution to the electrophysiology of human induced pluripotent stem cell-derived cardiomyocytes. Our results indicate that studies on isolated cells, out of tissue context, may underestimate the cellular ion channel properties being characterized. </div> <a href='#' class='show open-abstract' style='margin-left:10px;'>more »</a> <a href='#' class='hide close-abstract' style='margin-left:10px;'>« less</a> </div><div class="clearfix"></div> </div> </li> <li> <div class="article item document" itemscope itemtype="http://schema.org/TechArticle"> <div class="item-info"> <div class="title"> <a href="https://par.nsf.gov/biblio/10462444-integrated-modeling-framework-reveals-co-regulation-transcription-factors-mirnas-lncrnas-cardiac-developmental-dynamics" itemprop="url"> <span class='span-link' itemprop="name">Integrated modeling framework reveals co-regulation of transcription factors, miRNAs and lncRNAs on cardiac developmental dynamics</span> </a> </div> <div> <strong> <a class="misc external-link" href="https://doi.org/10.1186/s13287-023-03442-0" target="_blank" title="Link to document DOI">https://doi.org/10.1186/s13287-023-03442-0  <span class="fas fa-external-link-alt"></span></a> </strong> </div> <div class="metadata"> <span class="authors"> <span class="author" itemprop="author">Li, Shumin</span> <span class="sep">; </span><span class="author" itemprop="author">Yan, Bin</span> <span class="sep">; </span><span class="author" itemprop="author">Wu, Binbin</span> <span class="sep">; </span><span class="author" itemprop="author">Su, Junhao</span> <span class="sep">; </span><span class="author" itemprop="author">Lu, Jianliang</span> <span class="sep">; </span><span class="author" itemprop="author">Lam, Tak-Wah</span> <span class="sep">; </span><span class="author" itemprop="author">Boheler, Kenneth R.</span> <span class="sep">; </span><span class="author" itemprop="author">Poon, Ellen Ngar-Yun</span> <span class="sep">; </span><span class="author" itemprop="author">Luo, Ruibang</span> </span> <span class="year">( <time itemprop="datePublished" datetime="2023-09-13">September 2023</time> , Stem Cell Research & Therapy) </span> </div> <div style="cursor: pointer;-webkit-line-clamp: 5;" class="abstract" itemprop="description"> <title>Abstract Aims

Dissecting complex interactions among transcription factors (TFs), microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) are central for understanding heart development and function. Although computational approaches and platforms have been described to infer relationships among regulatory factors and genes, current approaches do not adequately account for how highly diverse, interacting regulators that include noncoding RNAs (ncRNAs) control cardiac gene expression dynamics over time.

 Methods

To overcome this limitation, we devised an integrated framework, cardiac gene regulatory modeling (CGRM) that integrates LogicTRN and regulatory component analysis bioinformatics modeling platforms to infer complex regulatory mechanisms. We then used CGRM to identify and compare the TF-ncRNA gene regulatory networks that govern early- and late-stage cardiomyocytes (CMs) generated by in vitro differentiation of human pluripotent stem cells (hPSC) and ventricular and atrial CMs isolated during in vivo human cardiac development.

 Results

Comparisons of in vitro versus in vivo derived CMs revealed conserved regulatory networks among TFs and ncRNAs in early cells that significantly diverged in late staged cells. We report that cardiac genes (“heart targets”) expressed in early-stage hPSC-CMs are primarily regulated by MESP1, miR-1, miR-23, lncRNAs NEAT1 and MALAT1, while GATA6, HAND2, miR-200c, NEAT1 and MALAT1 are critical for late hPSC-CMs. The inferred TF-miRNA-lncRNA networks regulating heart development and contraction were similar among early-stage CMs, among individual hPSC-CM datasets and between in vitro and in vivo samples. However, genes related to apoptosis, cell cycle and proliferation, and transmembrane transport showed a high degree of divergence between in vitro and in vivo derived late-stage CMs. Overall, late-, but not early-stage CMs diverged greatly in the expression of “heart target” transcripts and their regulatory mechanisms.

 Conclusions

In conclusion, we find that hPSC-CMs are regulated in a cell autonomous manner during early development that diverges significantly as a function of time when compared to in vivo derived CMs. These findings demonstrate the feasibility of using CGRM to reveal dynamic and complex transcriptional and posttranscriptional regulatory interactions that underlie cell directed versus environment-dependent CM development. These results with in vitro versus in vivo derived CMs thus establish this approach for detailed analyses of heart disease and for the analysis of cell regulatory systems in other biomedical fields.

 
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  • ; ; ; ; ; ; ; ( , Alcoholism: Clinical and Experimental Research)
    Background

    Alcohol use in pregnancy increases the risk of abnormal cardiac development, and excessive alcohol consumption in adults can induce cardiomyopathy, contractile dysfunction, and arrhythmias. Understanding molecular mechanisms underlying alcohol‐induced cardiac toxicity could provide guidance in the development of therapeutic strategies.

     Methods

    We have performed proteomic and bioinformatic analysis to examine protein alterations globally and quantitatively in cardiomyocytes derived from human‐induced pluripotent stem cells (hiPSC‐CMs) treated with ethanol (EtOH). Proteins in both cell lysates and extracellular culture media were systematically quantitated.

     Results

    Treatment with EtOH caused severe detrimental effects on hiPSC‐CMs as indicated by significant cell death and deranged Ca2+handling. Treatment of hiPSC‐CMs with EtOH significantly affected proteins responsible for stress response (e.g., GPX1 and HSPs), ion channel‐related proteins (e.g. ATP1A2), myofibril structure proteins (e.g., MYL2/3), and those involved in focal adhesion and extracellular matrix (e.g., ILK and PXN). Proteins involved in the TNF receptor‐associated factor 2 signaling (e.g., CPNE1 and TNIK) were also affected by EtOH treatment.

     Conclusions

    The observed changes in protein expression highlight the involvement of oxidative stress and dysregulation of Ca2+handling and contraction while also implicating potential novel targets in alcohol‐induced cardiotoxicity. These findings facilitate further exploration of potential mechanisms, discovery of novel biomarkers, and development of targeted therapeutics against EtOH‐induced cardiotoxicity.

     
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  • ; ; ; ; ( , Frontiers in Bioengineering and Biotechnology)
    Optogenetic methods for pacing of cardiac tissue can be realized by direct genetic modification of the cardiomyocytes to express light-sensitive actuators, such as channelrhodopsin-2, ChR2, or by introduction of light-sensitized non-myocytes that couple to the cardiac cells and yield responsiveness to optical pacing. In this study, we engineer three-dimensional “spark cells” spheroids, composed of ChR2-expressing human embryonic kidney cells (from 100 to 100,000 cells per spheroid), and characterize their morphology as function of cell density and time. These “spark-cell” spheroids are then deployed to demonstrate site-specific optical pacing of human stem-cell-derived cardiomyocytes (hiPSC-CMs) in 96-well format using non-localized light application and all-optical electrophysiology with voltage and calcium small-molecule dyes or genetically encoded sensors. We show that the spheroids can be handled using liquid pipetting and can confer optical responsiveness of cardiac tissue earlier than direct viral or liposomal genetic modification of the cardiomyocytes, with 24% providing reliable stimulation of the iPSC-CMs within 6 h and >80% within 24 h. Moreover, our data show that the spheroids can be frozen in liquid nitrogen for long-term storage and transportation, after which they can be deployed as a reagent on site for optical cardiac pacing. In all cases, optical stimulation was achieved at relatively low light levels (<0.15 mW/mm 2 ) when 5 ms or longer pulses were used. Our results demonstrate a scalable, cost-effective method with a cryopreservable reagent to achieve contactless optical stimulation of cardiac cell constructs without genetically modifying the myocytes, that can be integrated in a robotics-amenable workflow for high-throughput drug testing. 
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  • ; ; ; ; ; ; ; ; ; ; et al ( , Stem Cell Research & Therapy)
    Abstract Background

    Cardiac pathological outcome of metabolic remodeling is difficult to model using cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) due to low metabolic maturation.

     Methods

    hiPSC-CM spheres were treated with AMP-activated protein kinase (AMPK) activators and examined for hiPSC-CM maturation features, molecular changes and the response to pathological stimuli.

     Results

    Treatment of hiPSC-CMs with AMPK activators increased ATP content, mitochondrial membrane potential and content, mitochondrial DNA, mitochondrial function and fatty acid uptake, indicating increased metabolic maturation. Conversely, the knockdown of AMPK inhibited mitochondrial maturation of hiPSC-CMs. In addition, AMPK activator-treated hiPSC-CMs had improved structural development and functional features—including enhanced Ca2+transient kinetics and increased contraction. Transcriptomic, proteomic and metabolomic profiling identified differential levels of expression of genes, proteins and metabolites associated with a molecular signature of mature cardiomyocytes in AMPK activator-treated hiPSC-CMs. In response to pathological stimuli, AMPK activator-treated hiPSC-CMs had increased glycolysis, and other pathological outcomes compared to untreated cells.

     Conclusion

    AMPK activator-treated cardiac spheres could serve as a valuable model to gain novel insights into cardiac diseases.

     
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