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Title: An intrinsic, label-free signal for identifying stem cell-derived cardiomyocyte subtype
Abstract

Human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes have many promising applications, including the regeneration of injured heart muscles, cardiovascular disease modeling, and drug cardiotoxicity screening. Current differentiation protocols yield a heterogeneous cell population that includes pluripotent stem cells and different cardiac subtypes (pacemaking and contractile cells). The ability to purify these cells and obtain well-defined, controlled cell compositions is important for many downstream applications; however, there is currently no established and reliable method to identify hiPSC-derived cardiomyocytes and their subtypes. Here, we demonstrate that second harmonic generation (SHG) signals generated directly from the myosin rod bundles can be a label-free, intrinsic optical marker for identifying hiPSC-derived cardiomyocytes. A direct correlation between SHG signal intensity and cardiac subtype is observed, with pacemaker-like cells typically exhibiting ~70% less signal strength than atrial- and ventricular-like cardiomyocytes. These findings suggest that pacemaker-like cells can be separated from the heterogeneous population by choosing an SHG intensity threshold criteria. This work lays the foundation for developing an SHG-based high-throughput flow sorter for purifying hiPSC-derived cardiomyocytes and their subtypes.

</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 semi-colon-delimited-data"> <dt>Award ID(s):</dt> <dd> <span> <a target="_blank" rel="noopener noreferrer" href="https://par.nsf.gov/search/award_ids:1827611"> 1827611</a> </span> </dd> </dl> <dl class="dl-horizontal small"> <dt>NSF-PAR ID:</dt> <dd>10363223</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:"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:"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:"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:"Lieu, Deborah K.""><span class="author" itemprop="author">Lieu, Deborah K.</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></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="2019-12-09">2019-12-09</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>3</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. 390-394</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/10363338-human-induced-pluripotent-stem-cell-line-genetically-encoded-fluorescent-voltage-indicator-generated-via-crispr-action-potential-assessment-post-cardiogenesis" itemprop="url"> <span class='span-link' itemprop="name">Human induced pluripotent stem cell line with genetically encoded fluorescent voltage indicator generated via CRISPR for action potential assessment post-cardiogenesis</span> </a> </div> <div> <strong> <a class="misc external-link" href="https://doi.org/10.1002/stem.3085" target="_blank" title="Link to document DOI">https://doi.org/10.1002/stem.3085  <span class="fas fa-external-link-alt"></span></a> </strong> </div> <div class="metadata"> <span class="authors"> <span class="author" itemprop="author">Sun, Yao-Hui</span> <span class="sep">; </span><span class="author" itemprop="author">Kao, Hillary K. J.</span> <span class="sep">; </span><span class="author" itemprop="author">Chang, Che-Wei</span> <span class="sep">; </span><span class="author" itemprop="author">Merleev, Alexander</span> <span class="sep">; </span><span class="author" itemprop="author">Overton, James L.</span> <span class="sep">; </span><span class="author" itemprop="author">Pretto, Dalyir</span> <span class="sep">; </span><span class="author" itemprop="author">Yechikov, Sergey</span> <span class="sep">; </span><span class="author" itemprop="author">Maverakis, Emanual</span> <span class="sep">; </span><span class="author" itemprop="author">Chiamvimonvat, Nipavan</span> <span class="sep">; </span><span class="author" itemprop="author">Chan, James W.</span> <span class="sep">; </span><span class="author">et al</span></span> <span class="year">( <time itemprop="datePublished" datetime="2019-09-30">September 2019</time> , Stem Cells) </span> </div> <div style="cursor: pointer;-webkit-line-clamp: 5;" class="abstract" itemprop="description"> <title>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> </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/10455649-proteomic-profiling-reveals-roles-stress-response-ca-transient-dysregulation-novel-signaling-pathways-alcoholinduced-cardiotoxicity" itemprop="url"> <span class='span-link' itemprop="name">Proteomic Profiling Reveals Roles of Stress Response, Ca 2+ Transient Dysregulation, and Novel Signaling Pathways in Alcohol‐Induced Cardiotoxicity</span> </a> </div> <div> <strong> <a class="misc external-link" href="https://doi.org/10.1111/acer.14471" target="_blank" title="Link to document DOI">https://doi.org/10.1111/acer.14471  <span class="fas fa-external-link-alt"></span></a> </strong> </div> <div class="metadata"> <span class="authors"> <span class="author" itemprop="author">Liu, Rui</span> <span class="sep">; </span><span class="author" itemprop="author">Sun, Fangxu</span> <span class="sep">; </span><span class="author" itemprop="author">Forghani, Parvin</span> <span class="sep">; </span><span class="author" itemprop="author">Armand, Lawrence C.</span> <span class="sep">; </span><span class="author" itemprop="author">Rampoldi, Antonio</span> <span class="sep">; </span><span class="author" itemprop="author">Li, Dong</span> <span class="sep">; </span><span class="author" itemprop="author">Wu, Ronghu</span> <span class="sep">; </span><span class="author" itemprop="author">Xu, Chunhui</span> </span> <span class="year">( <time itemprop="datePublished" datetime="2020-10-16">October 2020</time> , Alcoholism: Clinical and Experimental Research) </span> </div> <div style="cursor: pointer;-webkit-line-clamp: 5;" class="abstract" itemprop="description"> <sec><title>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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  • 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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  • null (Ed.)
    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. 
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  • Abstract

    Efficient generation of cardiomyocytes from human-induced pluripotent stem cells (hiPSCs) is important for their application in basic and translational studies. Space microgravity can significantly change cell activities and function. Previously, we reported upregulation of genes associated with cardiac proliferation in cardiac progenitors derived from hiPSCs that were exposed to space microgravity for 3 days. Here we investigated the effect of long-term exposure of hiPSC-cardiac progenitors to space microgravity on global gene expression. Cryopreserved 3D hiPSC-cardiac progenitors were sent to the International Space Station (ISS) and cultured for 3 weeks under ISS microgravity and ISS 1 G conditions. RNA-sequencing analyses revealed upregulation of genes associated with cardiac differentiation, proliferation, and cardiac structure/function and downregulation of genes associated with extracellular matrix regulation in the ISS microgravity cultures compared with the ISS 1 G cultures. Gene ontology analysis and Kyoto Encyclopedia of Genes and Genomes mapping identified the upregulation of biological processes, molecular function, cellular components, and pathways associated with cell cycle, cardiac differentiation, and cardiac function. Taking together, these results suggest that space microgravity has a beneficial effect on the differentiation and growth of cardiac progenitors.

     
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