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1. A computational model of induced pluripotent stem‐cell derived cardiomyocytes incorporating experimental variability from multiple data sources

   https://doi.org/10.1113/JP277724  

   Kernik, Divya C. ; Morotti, Stefano ; Wu, HaoDi ; Garg, Priyanka ; Duff, Henry J. ; Kurokawa, Junko ; Jalife, José ; Wu, Joseph C. ; Grandi, Eleonora ; Clancy, Colleen E. ( July 2019 , The Journal of Physiology)

   Induced pluripotent stem cell‐derived cardiomyocytes (iPSC‐CMs) capture patient‐specific genotype–phenotype relationships, as well as cell‐to‐cell variability of cardiac electrical activity

   Computational modelling and simulation provide a high throughput approach to reconcile multiple datasets describing physiological variability, and also identify vulnerable parameter regimes

   We have developed a whole‐cell model of iPSC‐CMs, composed of single exponential voltage‐dependent gating variable rate constants, parameterized to fit experimental iPSC‐CM outputs

   We have utilized experimental data across multiple laboratories to model experimental variability and investigate subcellular phenotypic mechanisms in iPSC‐CMs

   This framework links molecular mechanisms to cellular‐level outputs by revealing unique subsets of model parameters linked to known iPSC‐CM phenotypes

   There is a profound need to develop a strategy for predicting patient‐to‐patient vulnerability in the emergence of cardiac arrhythmia. A promisingin vitromethod to address patient‐specific proclivity to cardiac disease utilizes induced pluripotent stem cell‐derived cardiomyocytes (iPSC‐CMs). A major strength of this approach is that iPSC‐CMs contain donor genetic information and therefore capture patient‐specific genotype–phenotype relationships. A cited detriment of iPSC‐CMs is the cell‐to‐cell variability observed in electrical activity. We postulated, however, that cell‐to‐cell variability may constitute a strength when appropriately utilized in a computational framework to build cell populations that can be employed to identify phenotypic mechanisms and pinpoint key sensitive parameters. Thus, we have exploited variation in experimental data across multiple laboratories to develop a computational framework for investigating subcellular phenotypic mechanisms. We have developed a whole‐cell model of iPSC‐CMs composed of simple model components comprising ion channel models with single exponential voltage‐dependent gating variable rate constants, parameterized to fit experimental iPSC‐CM data for all major ionic currents. By optimizing ionic current model parameters to multiple experimental datasets, we incorporate experimentally‐observed variability in the ionic currents. The resulting population of cellular models predicts robust inter‐subject variability in iPSC‐CMs. This approach links molecular mechanisms to known cellular‐level iPSC‐CM phenotypes, as shown by comparing immature and mature subpopulations of models to analyse the contributing factors underlying each phenotype. In the future, the presented models can be readily expanded to include genetic mutations and pharmacological interventions for studying the mechanisms of rare events, such as arrhythmia triggers.

    

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2. Integration of Engineered “Spark-Cell” Spheroids for Optical Pacing of Cardiac Tissue

   https://doi.org/10.3389/fbioe.2021.658594  

   Chua, Christianne J. ; Han, Julie L. ; Li, Weizhen ; Liu, Wei ; Entcheva, Emilia ( June 2021 , 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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3. Human induced pluripotent stem cell line with genetically encoded fluorescent voltage indicator generated via CRISPR for action potential assessment post-cardiogenesis

   https://doi.org/10.1002/stem.3085  

   Sun, Yao-Hui ; Kao, Hillary K. J. ; Chang, Che-Wei ; Merleev, Alexander ; Overton, James L. ; Pretto, Dalyir ; Yechikov, Sergey ; Maverakis, Emanual ; Chiamvimonvat, Nipavan ; Chan, James W. ; et al ( September 2019 , Stem Cells)

   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.

   Producing mature and functional cardiomyocytes (CMs) by in vitro differentiation of induced pluripotent stem cells (iPSCs) using only biochemical cues is challenging. To mimic the biophysical and biomechanical complexity of the native in vivo environment during the differentiation and maturation process, polydimethylsiloxane substrates with 3D topography at the micrometer and sub‐micrometer levels are developed and used as cell‐culture substrates. The results show that while cylindrical patterns on the substrates resembling mature CMs enhance the maturation of iPSC‐derived CMs, sub‐micrometer‐level topographical features derived by imprinting primary human CMs further accelerate both the differentiation and maturation processes. The resulting CMs exhibit a more‐mature phenotype than control groups—as confirmed by quantitative polymerase chain reaction, flow cytometry, and the magnitude of beating signals—and possess the shape and orientation of mature CMs in human myocardium—as revealed by fluorescence microscopy, Ca²⁺flow direction, and mitochondrial distribution. The experiments, combined with a virtual cell model, show that the physico‐mechanical cues generated by these 3D‐patterned substrates improve the phenotype of the CMs via the reorganization of the cytoskeletal network and the regulation of chromatin conformation.

    

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4. Syncytium cell growth increases Kir2.1 contribution in human iPSC-cardiomyocytes

   https://doi.org/10.1152/ajpheart.00148.2020  

   Li, Weizhen ; Han, Julie L. ; Entcheva, Emilia ( November 2020 , American Journal of Physiology-Heart and Circulatory Physiology)

   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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