We thank Blake Wu for proofreading the manuscript. This work was supported by the National Institutes of Health (NIH) R01 HL113006, R01 HL141371, R01 HL163680, R01 HL141851, U01FD005978, and NASA NNX16A069A (JCW), and AHA Postdoctoral Fellowship 872244 (GMP).

1. Preparation of media and solutions
<ol>
	<li>Prepare hiPSC-CM maintenance medium by mixing a 10 mL bottle of 50x B27 supplement and 500 mL of RPMI 1640 medium. Store the medium at 4 &#176;C and use it within a month. Equilibrate the medium to room temperature (RT) before use.</li>
	<li>Prepare hiPSC-CM seeding medium by mixing 20 mL of serum replacement and 180 mL of hiPSC-CM maintenance medium (10% dilution, v/v). While freshly prepared seeding medium is preferred, it can be stored at 4 &#176;C for no more than 2 weeks. Equilibrate the medium to RT before use.</li>
	<li>Prepare extracellular matrix coating solution by thawing one bottle (10 mL) of basement membrane matrix at 4 &#176;C overnight and aliquoting 500 &#181;L of basement membrane matrix in sterile 1.5 mL tubes. Store at -20 &#176;C for further use. Mix 500 &#181;L of basement membrane matrix and 100 mL of ice-cold DMEM/F12 medium (1:200 dilution, v/v) to make basement membrane matrix coating solution.</li>
	<li>Prepare 50 mL of Tyrode&#39;s solution containing 135 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM glucose, and 10 mM HEPES. Adjust the pH to 7.4 with NaOH. Prepare fresh Tyrode&#39;s solution on the day of the experiment.</li>
	<li>Prepare 50 mL of intracellular pipette solution containing 120 mM KCl, 1 mM MgCl2, 10 mM EGTA, and 10 mM HEPES. Adjust the pH to 7.2 with KOH. Aliquot the intracellular pipette solution in sterile 5 mL tubes and store it at -20 &#176;C. On the day of the experiment, freshly add MgATP to the solution to achieve a final concentration of 3 mM.</li>
	<li>Prepare 1 mL of Fura-2 AM loading solution containing 2 &#181;M Fura-2 AM and 0.1% Pluronic F-127. Prepare fresh loading solution on the day of the experiment and protect it from the light.</li>
</ol>
2. Measurement of hiPSC-CM contraction motion
<ol>
	<li>Prepare basement membrane matrix-coated plates by adding 100 &#181;L of extracellular matrix coating solution to each well of 96-well plates. Incubate the 96-well plates in a humidified cell culture incubator at 37 &#176;C, 5% CO2 overnight.</li>
	<li>Enzymatic dissociation of hiPSC-CMs
	<ol>
		<li>Request hiPSCs from Stanford Cardiovascular Institute iPSC Biobank&#160;(http://med.stanford.edu/scvibiobank.html). Generate hiPSC-CMs according to the previous publications8,9. Observe the hiPSC-CMs cultured in six-well plates under a microscope at 10x magnification. 
		NOTE: Do not dissociate the cells when they are still proliferative. Otherwise, the cells will overgrow on the wells after replating and lead to inaccurate results of the functional assays. Usually, hiPSC-CMs stop proliferation at days 18-23.</li>
		<li>Make sure the hiPSC-CMs are beating strongly and have a purity of over 95%. Assess the purity by immunostaining against cardiac troponin T, a specific marker of cardiomyocytes8,9.</li>
		<li>Aspirate the maintenance medium and wash the cells with 1x DPBS twice. Add 1 mL of cell detachment solution to each well of the six-well plates and incubate for 10 min at 37 &#176;C. 
		NOTE: The incubation duration should be regularly monitored. Make sure not to over-digest the cells as it will reduce cell viability.</li>
		<li>Gently pipette hiPSC-CMs up and down several times using a 5 mL pipette to generate a single-cell suspension. Add an equal volume of hiPSC-CMs seeding medium to neutralize the cell detachment solution.</li>
		<li>Centrifuge hiPSC-CMs at 300 x g for 3 min at RT. Aspirate the supernatant and resuspend the cell pellet in 1-2 mL of hiPSC-CM seeding medium.</li>
		<li>Quantify the cell density and viability using a trypan blue exclusion method. Briefly, mix 10 &#181;L of cell suspension with an equal volume of trypan blue, transfer to a cell counting slide, and then count using a cell counter, as previously described21.</li>
	</ol>
	</li>
	<li>Seeding hiPSC-CMs
	<ol>
		<li>Remove the extracellular matrix coating solution from the 96-well plates.</li>
		<li>Seed 50,000 cells per well in 100 &#181;L of hiPSC-CM seeding medium and place the 96-well plates in a humidified cell culture incubator at 37 &#176;C, 5% CO2 overnight.</li>
		<li>Replace the hiPSC-CM seeding medium with 100 &#181;L of hiPSC-CM maintenance medium 1 day after plating. Change the hiPSC-CM maintenance medium every 2 days until recording.</li>
	</ol>
	</li>
	<li>Data acquisition
	<ol>
		<li>Measure the hiPSC-CM contraction motion at least 10 days post-plating (recommended).</li>
		<li>Turn on the temperature controller and set 37 &#176;C as the preferred temperature. Turn on the microscope, camera, and CO2 supply.</li>
		<li>Fill the water jacket of the plate holder with an appropriate amount of sterile deionized water (Figure 2A). Place the 96-well plate on the plate holder and allow cells to acclimate for at least 5 min.</li>
		<li>Open the view software, set the camera frame rate to 75 frames per second (fps) and the video/image resolution to 1024 &#215; 1024 pixels. Apply the auto-focus and auto-brightness functions. Record videos and images of hiPSC-CMs. 
		NOTE: Avoid vibration of the microscope table during recording.</li>
		<li>Open the analyzer software for automatic analysis.</li>
	</ol>
	</li>
</ol>
3. Measurement of hiPSC-CM field potential
<ol>
	<li>Preparation of basement membrane matrix-coated plates
	<ol>
		<li>Carefully place an 8 &#181;L droplet of extracellular matrix coating solution over the recording electrode area of each well of the 48-well microelectrode array (MEA) plates. See Figure 3A for the appropriate electrode area for droplet placement. This step is critical to keeping the hiPSC-CM monolayer concentrated on the electrodes. Avoid touching the electrodes in any circumstances.</li>
		<li>Add 3 mL of sterile deionized water to the area surrounding the wells (Figure 3A) of the 48-well MEA plates to prevent coating solution evaporation. Incubate 48-well MEA plates in a humidified cell culture incubator at 37 &#176;C, 5% CO2 for 45-60 min. 
		NOTE: Do not incubate for too long to prevent the basement membrane matrix from drying.</li>
	</ol>
	</li>
	<li>Perform enzymatic dissociation of hiPSC-CMs as described in step 2.2.</li>
	<li>Seeding hiPSC-CMs
	<ol>
		<li>Remove most of the extracellular matrix medium from the well surface without touching the electrodes using a 200 &#181;L pipette tip within the same single row or column.</li>
		<li>Seed 50,000 cells per well in an 8 &#181;L droplet of hiPSC-CM seeding medium over the recording electrode area of each well of the same row or column.</li>
		<li>Repeat the last two steps until all wells have been plated with cells. Check under the microscope that all wells have the hiPSC-CM suspension. For the desired density, see Figure 3B. 
		NOTE: Attachment of hiPSC-CMs will be compromised if the basement membrane matrix is completely dried out, making cell attachment suboptimal.</li>
		<li>Incubate the 48-well MEA plates in a humidified cell culture incubator at 37 &#176;C, 5% CO2 for 60 min.</li>
		<li>Slowly and gently add 150 &#181;L of hiPSC-CM seeding medium to each well of the 48-well MEA plates.To add medium without dispersing previously seeded hiPSC-CMs, hold the plate at a 45&#176; angle, lean the pipette tip against the wall of each well, and release the medium slowly. 
		NOTE: Adding medium too quickly or with high pressure will dislodge the adhered cardiomyocytes. Avoid direct contact with the hiPSC-CM drop suspension.</li>
		<li>Incubate the 48-well MEA plates in a humidified cell culture incubator at 37 &#176;C, 5% CO2 overnight.</li>
		<li>Refresh the hiPSC-CM seeding medium with 300 &#181;L of hiPSC-CM maintenance medium 1 day after plating. Change the hiPSC-CM maintenance medium every 2 days before recording.</li>
	</ol>
	</li>
	<li>Data acquisition
	<ol>
		<li>Perform MEA measurement at least 10 days post-plating (recommended). On day 10 post-plating, check the density of spontaneous beating hiPSC-CM monolayer under a microscope at 10x magnification, which should be as shown in Figure 3C.</li>
		<li>Place the 48-well MEA plate in the recording instrument. The touch screen allows to observe the temperature (37 &#176;C) and CO2 (5%) status.</li>
		<li>Open the navigator software. In the experimental setup window, select Cardiac Real-time Configuration and Field Potential Recordings. Apply spontaneous or paced beating configurations.</li>
		<li>In beat detection parameters, set detection threshold to 300 &#181;V, minimum beating period to 250 ms, and maximum beating period to 5 s. Select Polynomial Regression for field potential duration (FPD) calculation.</li>
		<li>Check whether the baseline electrical activity signal is mature and stable. 
		NOTE: The mature waveforms recorded by each electrode from the multi-well MEA plate must reflect the cardiac field potential with easily identifiable characteristics coinciding with the cardiac depolarization spike and repolarization phase, as shown in Figure 3D-F.</li>
		<li>Acquire the baseline cardiac activities for 1-3 min. If drug treatment is needed, add compounds to the wells after baseline recording. Place the plate in an incubator for at least 30 min before the next recording.</li>
	</ol>
	</li>
</ol>
4. Measurement of hiPSC-CM action potential
<ol>
	<li>Prepare basement membrane matrix-coated dishes by placing 500 &#181;L of extracellular matrix coating solution on the glass-bottom part of 35 mm dishes (Figure 4A). Incubate the 35 mm dishes in a humidified cell culture incubator at 37 &#176;C, 5% CO2 overnight.</li>
	<li>Perform enzymatic dissociation of hiPSC-CMs as described in step 2.2.</li>
	<li>Seeding hiPSC-CMs
	<ol>
		<li>Remove the extracellular matrix coating solution from the 35 mm dishes. Seed 50,000 cells per well in 500 &#181;L of hiPSC-CM seeding medium on the glass bottom part of 35 mm dishes.</li>
		<li>Incubate the 35 mm dishes in a humidified cell culture incubator at 37 &#176;C, 5% CO2 overnight. Remove the hiPSC-CM seeding medium and add 2 mL of hiPSC-CM maintenance medium to the 35 mm dishes. 
		NOTE: It is recommended to put a 200 &#181;L non-filtered tip over the 2 mL aspiration pipette to remove the spent medium to avoid touching cells.</li>
		<li>Change the hiPSC-CM culture medium every 2 days before recording.</li>
	</ol>
	</li>
	<li>Data acquisition
	<ol>
		<li>Perform action potential measurement at least 10 days post-plating (recommended). Prepare fresh Tyrode&#39;s solution as described in step 1.4. Thaw one aliquot of intracellular pipette solution and freshly add MgATP to a final concentration of 3 mM.</li>
		<li>Pull the micropipettes from borosilicate glass capillaries using a micropipette puller. 
		NOTE: A resistance of 2-5 M&#937; of the micropipettes is preferred.</li>
		<li>Replace the hiPSC-CM maintenance medium with Tyrode&#39;s solution and allow the cells to acclimate to Tyrode&#39;s solution for 15 min (Figure 4C). Insert the temperature sensors in the chamber, put the 35 mm dish in the chamber as shown in Figure 4C, and adjust the temperature to 37 &#176;C (Figure 4D).</li>
		<li>Fill the micropipettes with intracellular solution and insert them into the holder connected to the patch-clamp amplifier headstage (Figure 4E). Open the stimulation/acquisition software, load the action potential recording protocol, and select the voltage-clamp configuration.</li>
		<li>Insert the micropipette into the bath solution, select appropriate single hiPSC-CMs, and adjust and lower the position of the micropipette next to the selected cell using a micromanipulator. When the pipette tip is inserted into the bath solution, check for pipette resistance that can be observed on the oscilloscope monitor.</li>
		<li>Position the pipette close to the cell, and the current pulses will decrease slightly to reflect the increasing seal resistance. Apply gentle suction with negative pressure to increase the resistance, which will form a gigaseal. Apply the Seal function.</li>
		<li>Apply additional suction to break the membrane to get a whole-cell recording configuration. Once the membrane portion within the pipette area is ruptured by suction, the internal solution is in equilibrium with the cell cytoplasm. At this point, switch from voltage-clamp mode (V-clamp) to current-clamp mode or no current injection (C-Clamp) using the amplifier dialog. Press the Record button to generate and save the action potential recording files. 
		NOTE: The mode change provides a continuous recording of the spontaneous action potentials of hiPSC-CMs via no trigger and no stimulation recording protocol, which can be monitored at the V-mon output of the patch-clamp amplifier.</li>
	</ol>
	</li>
</ol>
5. Measurement of hiPSC-CM Ca2+ transient
<ol>
	<li>Refer to the previous protocol for preparing customized cell chambers for Ca2+ imaging21. For the preparation of the basement membrane matrix-coated cell chamber, place 200 &#181;L of extracellular matrix coating solution in the cell chambers. Incubate the cell chambers in a humidified cell culture incubator at 37 &#176;C, 5% CO2 overnight.</li>
	<li>Perform enzymatic dissociation of hiPSC-CMs as described in step 2.2.</li>
	<li>Seeding hiPSC-CMs
	<ol>
		<li>Remove the extracellular matrix coating solution from the cell chambers. Seed 20,000 cells per well in 200 &#181;L of hiPSC-CM seeding medium.</li>
		<li>Replace the hiPSC-CM seeding medium with 200 &#181;L of hiPSC-CM maintenance medium 1 day after plating. Change the hiPSC-CM maintenance medium every 2 days before recording.</li>
	</ol>
	</li>
	<li>Data acquisition
	<ol>
		<li>Perform Ca2+ transient measurement at least 10 days post-plating (recommended). Prepare fresh Tyrode&#39;s solution as described in step 1.4. Prepare fresh Fura-2 AM loading solution as described in step 1.6.</li>
		<li>Aspirate the hiPSC-CM maintenance medium and wash with Tyrode&#39;s solution twice. Apply 100 &#181;L of Fura-2 AM loading solution and incubate for 10 min at RT.</li>
		<li>Replace the Fura-2 AM loading solution with Tyrode&#39;s solution and allow at least 5 min for complete de-esterification of Fura-2 AM.</li>
		<li>Add a drop of immersion oil on the 40x objective of an inverted epifluorescence microscope equipped with a 40x oil immersion objective (0.95 NA). Place the cell chamber in the stage adapter of the microscope (Figure 5A).</li>
		<li>Install the temperature controller wires to the bath chamber and set it to 37 &#176;C. Install the electrical field stimulation electrodes and set the pulses at 0.5 Hz with a 20 ms duration.</li>
		<li>Turn on the ultra-high-speed wavelength-switching light source and set it to 340 nm and 380 nm fast-switching mode according to the instrument&#39;s instructions.</li>
		<li>Apply an exposure time of 20 ms for both wavelengths and a recording time of 20 s in the software. Record the video reflecting real-time changes of Ca2+ transient in hiPSC-CMs. Export the data to a spreadsheet and analyze the data as previously described23.</li>
	</ol>
	</li>
</ol>

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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+the+transcriptome+signatures+of+calcium+channel+blockers+in+human+induced+pluripotent+stem+cell-derived+cardiomyocytes.">Identifying the transcriptome signatures of calcium channel blockers in human induced pluripotent stem cell-derived cardiomyocytes.</a> Circulation Research. 125 (2), 212-222 (2019).</li><li>Seeger, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+premature+termination+codon+mutation+in+MYBPC3+causes+hypertrophic+cardiomyopathy+via+chronic+activation+of+nonsense-mediated+decay.">A premature termination codon mutation in MYBPC3 causes hypertrophic cardiomyopathy via chronic activation of nonsense-mediated decay.</a> Circulation. 139 (6), 799-811 (2019).</li><li>Zhang, J. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cryopreservation+on+human+induced+pluripotent+stem+cell-derived+cardiomyocytes+for+assessing+drug+safety+response+profiles.">Effects of cryopreservation on human induced pluripotent stem cell-derived cardiomyocytes for assessing drug safety response profiles.</a> Stem Cell Reports. 16 (1), 168-181 (2021).</li><li>Yu, B., Zhao, S. R., Yan, C. D., Zhang, M., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deconvoluting+the+cells+of+the+human+heart+with+iPSC+technology:+cell+types,+protocols,+and+uses.">Deconvoluting the cells of the human heart with iPSC technology: cell types, protocols, and uses.</a> Current Cardiology Reports. 24 (5), 487-496 (2022).</li><li>Thomas, D., Cunningham, N. J., Shenoy, S., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human-induced+pluripotent+stem+cells+in+cardiovascular+research:+current+approaches+in+cardiac+differentiation,+maturation+strategies,+and+scalable+production.">Human-induced pluripotent stem cells in cardiovascular research: current approaches in cardiac differentiation, maturation strategies, and scalable production.</a> Cardiovascular Research. 118 (1), 20-36 (2022).</li><li>Zhu, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+developmental+cues+for+the+maturation+of+human+pluripotent+stem+cell-derived+cardiomyocytes.">Physical developmental cues for the maturation of human pluripotent stem cell-derived cardiomyocytes.</a> Stem Cell Research &amp; Therapy. 5 (5), 117 (2014).</li><li>Feyen, D. A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+maturation+media+improve+physiological+function+of+human+iPSC-derived+cardiomyocytes.">Metabolic maturation media improve physiological function of human iPSC-derived cardiomyocytes.</a> Cell Reports. 32 (3), 107925 (2020).</li><li>Gintant, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+human+induced+pluripotent+stem+cell-derived+cardiomyocytes+in+preclinical+cancer+drug+cardiotoxicity+testing:+a+scientific+statement+from+the+American+Heart+Association.">Use of human induced pluripotent stem cell-derived cardiomyocytes in preclinical cancer drug cardiotoxicity testing: a scientific statement from the American Heart Association.</a> Circulation Research. 125 (10), 75-92 (2019).</li><li>Kussauer, S., David, R., Lemcke, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=hiPSCs+Derived+cardiac+cells+for+drug+and+toxicity+screening+and+disease+modeling:+what+micro-+electrode-array+analyses+can+tell+us.">hiPSCs Derived cardiac cells for drug and toxicity screening and disease modeling: what micro- electrode-array analyses can tell us.</a> Cells. 8 (11), (2019).</li><li>Ng, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+phenotyping+of+heteromeric+human+ether-a-go-go-related+gene+potassium+channel+variants+can+discriminate+pathogenic+from+rare+benign+variants.">High-throughput phenotyping of heteromeric human ether-a-go-go-related gene potassium channel variants can discriminate pathogenic from rare benign variants.</a> Heart Rhythm. 17 (3), 492-500 (2020).</li><li>Obergrussberger, A., Friis, S., Bruggemann, A., Fertig, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+patch+clamp+in+drug+discovery:+major+breakthroughs+and+innovation+in+the+last+decade.">Automated patch clamp in drug discovery: major breakthroughs and innovation in the last decade.</a> Expert Opinion on Drug Discovery. 16 (1), 1-5 (2021).</li><li>Kozek, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+discovery+of+trafficking-deficient+variants+in+the+cardiac+potassium+channel+KV11.1.">High-throughput discovery of trafficking-deficient variants in the cardiac potassium channel KV11.1.</a> Heart Rhythm. 17 (12), 2180-2189 (2020).</li><li>Heyne, H. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+functional+effects+of+missense+variants+in+voltage-gated+sodium+and+calcium+channels.">Predicting functional effects of missense variants in voltage-gated sodium and calcium channels.</a> Science Translational Medicine. 12 (556), (2020).</li><li>Li, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+an+automated+patch-clamp+platform+for+electrophysiological+and+pharmacological+evaluation+of+hiPSC-CMs.">Establishment of an automated patch-clamp platform for electrophysiological and pharmacological evaluation of hiPSC-CMs.</a> Stem Cell Research. 41, 101662 (2019).</li><li>Li, W., Luo, X., Ulbricht, Y., Guan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blebbistatin+protects+iPSC-CMs+from+hypercontraction+and+facilitates+automated+patch-clamp+based+electrophysiological+study.">Blebbistatin protects iPSC-CMs from hypercontraction and facilitates automated patch-clamp based electrophysiological study.</a> Stem Cell Research. 56, 102565 (2021).</li><li>Milligan, C. J., M&#246;ller, C., Gamper, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ion Channels: Methods and Protocols. , 171-187 (2013).</li></ol>

J.C.W. is a co-founder of Greenstone Biosciences but has no competing interests, as the work presented here is completely independent. The other authors declare no competing interests.

Human iPSC technology has emerged as a powerful platform for disease modeling and drug screening. Here, we describe a detailed protocol for measuring hiPSC-CM contractility, field potential, action potential, and Ca2+ transient. This protocol provides a comprehensive characterization of hiPSC-CM contractility and electrophysiology. These functional assays have been applied in multiple publications from our group12,13,18</su...

Drug development is a long and expensive process. A study of new therapeutic drugs approved by the US Food and Drug Administration (FDA) between 2009 and 2018 reported that the estimated median cost of capitalized research and clinical trials was $985 million per product1. Drug-induced cardiotoxicity is the leading cause of drug attrition and withdrawal from the market2. Notably, cardiotoxicity is reported among multiple classes of therapeutic drugs3. Therefore, cardiac safety assessment is a crucial component during the drug development process. The current paradigm for cardiac safety assessment is still highly dependent on animal models. However, species differences from the use of animal models are increasingly recognized as a primary cause of inaccurate predictions for drug-induced cardiotoxicity in human patients4. For example, the morphology of cardiac action potential differs substantially between humans and mice due to the contributions from different repolarizing currents5. In addition, differential isoforms of cardiac myosin and circular RNAs that can impact cardiac physiology have been well documented among species6,7. To bridge these gaps, it is imperative to establish a reliable, efficient, and human-based model for preclinical cardiac safety assessment.
The groundbreaking invention of induced pluripotent stem cell (iPSC) technology has generated unprecedented drug screening and disease modeling platforms. Over the past decade, methods to generate human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become well established8,9. hiPSC-CMs have attracted great interest in their potential applications in disease modeling, drug-induced cardiotoxicity screening, and precision medicine. For instance, hiPSC-CMs have been utilized to model the pathologic phenotypes of cardiac diseases caused by genetic inheritance, such as long QT syndrome10, hypertrophic cardiomyopathy11,12, and dilated cardiomyopathy13,14,15. Consequently, key signaling pathways implicated in the pathogenesis of cardiac diseases have been identified, which can shed light on potential therapeutic strategies for effective treatment. Moreover, hiPSC-CMs have been used to screen drug-induced cardiotoxicity associated with anticancer agents, including doxorubicin, trastuzumab, and tyrosine kinase inhibitors16,17,18; strategies to mitigate the resultant cardiotoxicity are under investigation. Finally, the genetic information retained in hiPSC-CMs allows for the screening and prediction of drug-induced cardiotoxicity at both individual and population levels19,20. Collectively, hiPSC-CMs have proven to be an invaluable tool for personalized cardiac safety prediction.
The overall goal of this protocol is to establish methodologies to comprehensively and efficiently investigate the functional characteristics of hiPSC-CMs, which are of great importance in applying hiPSC-CMs toward disease modeling, drug-induced cardiotoxicity screening, and precision medicine. Here, we detail an array of functional assays to assess the functional properties of hiPSC-CMs, including the measurement of contractility, field potential, action potential, and calcium (Ca2+) handling (Figure 1).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>35 mm glass bottom dish with 20 mm micro-well #1.5 cover glass</td>
			<td>Cellvis</td>
			<td>D35-20-1.5-N</td>
			<td>Patch clamp</td>
		</tr>
		<tr>
			<td>50x B27 supplements</td>
			<td>Life Technologies</td>
			<td>17504-044</td>
			<td>hiPSC-CM culture medium</td>
		</tr>
		<tr>
			<td>6-well culture plate</td>
			<td>E &#38; K Scientific</td>
			<td>EK-27160</td>
			<td>hiPSC-CM culture</td>
		</tr>
		<tr>
			<td>96-well flat clear bottom black polystyrene TC-treated microplates</td>
			<td>Corning</td>
			<td>3603</td>
			<td>Contraction motion measurement</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Sigma-Aldrich</td>
			<td>A6964</td>
			<td>Enzymatic dissociation</td>
		</tr>
		<tr>
			<td>Axion&#39;s Integrated Studio (AxIS)</td>
			<td>Axion Biosystems</td>
			<td></td>
			<td>navigator software</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries</td>
			<td>Harvard Apparatus</td>
			<td>BF 100-50-10,</td>
			<td>Patch clamp</td>
		</tr>
		<tr>
			<td>CaCl2 1 M in H2O</td>
			<td>Sigma-Aldrich</td>
			<td>21115</td>
			<td>Tyrode&#8217;s solution</td>
		</tr>
		<tr>
			<td>Cell counting chamber slides</td>
			<td>ThermoFisher Scientific</td>
			<td>C10228</td>
			<td>Cell counting</td>
		</tr>
		<tr>
			<td>CytoView 48-well MEA plates</td>
			<td>Axion Biosystems</td>
			<td>M768-tMEA-48B</td>
			<td>MEA</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco/Life Technologies</td>
			<td>12634028</td>
			<td>Extracellular matrix medium</td>
		</tr>
		<tr>
			<td>DPBS, no calcium, no magnesium</td>
			<td>Fisher Scientific</td>
			<td>14-190-250</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>E3889</td>
			<td>Intracellular pipette solution</td>
		</tr>
		<tr>
			<td>EPC 10 USB&#160;patch clamp amplifier</td>
			<td>Warner Instruments</td>
			<td>89-5000</td>
			<td>Patch clamp</td>
		</tr>
		<tr>
			<td>Fura-2, AM, cell permeant</td>
			<td>ThermoFisher Scientific</td>
			<td>F1221</td>
			<td>Ca2+ transient measurement</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td>Tyrode&#8217;s solution</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td>Tyrode&#8217;s solution</td>
		</tr>
		<tr>
			<td>hiPSCs</td>
			<td>Stanford Cardiovascular Institute iPSC Biobank</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>529552</td>
			<td>Tyrode&#8217;s solution</td>
		</tr>
		<tr>
			<td>KnockOut Serum Replacement</td>
			<td>ThermoFisher Scientific</td>
			<td>10828-028</td>
			<td>hiPSC-CM seeding medium</td>
		</tr>
		<tr>
			<td>KOH 8&#160;M</td>
			<td>Sigma-Aldrich</td>
			<td>P4494</td>
			<td>Intracellular pipette solution</td>
		</tr>
		<tr>
			<td>Lambda DG 4</td>
			<td>Sutter Instrument Company</td>
			<td></td>
			<td>Ca2+ transient measurement; ultra-high-speed wavelength switching light source</td>
		</tr>
		<tr>
			<td>Luna-FL automated fluorescence cell counter</td>
			<td>WISBIOMED</td>
			<td>LB-L20001</td>
			<td>Cell counting</td>
		</tr>
		<tr>
			<td>Maestro Pro&#160;MEA system</td>
			<td>Axion Biosystems</td>
			<td></td>
			<td>MEA</td>
		</tr>
		<tr>
			<td>Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix</td>
			<td>Corning</td>
			<td>356231</td>
			<td>Extracellular matrix medium</td>
		</tr>
		<tr>
			<td>MgATP</td>
			<td>Sigma-Aldrich</td>
			<td>A9187</td>
			<td>Intracellular pipette solution</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td>Tyrode&#8217;s solution</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td>Tyrode&#8217;s solution</td>
		</tr>
		<tr>
			<td>NaOH 10&#160;M</td>
			<td>Sigma-Aldrich</td>
			<td>72068</td>
			<td>Tyrode&#8217;s solution</td>
		</tr>
		<tr>
			<td>NIS Elements AR</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127 (20% Solution in DMSO)</td>
			<td>ThermoFisher Scientific</td>
			<td>P3000MP</td>
			<td>Ca2+ transient measurement</td>
		</tr>
		<tr>
			<td>RPMI 1640 medium</td>
			<td>Life Technologies</td>
			<td>11875-119</td>
			<td>hiPSC-CM culture medium</td>
		</tr>
		<tr>
			<td>Sony SI8000 Cell Motion Imaging System</td>
			<td>Sony Biotechnology</td>
			<td></td>
			<td>Contraction motion measurement</td>
		</tr>
		<tr>
			<td>Sutter Micropipette puller</td>
			<td>Sutter Instruments</td>
			<td>P-97</td>
			<td>Patch clamp</td>
		</tr>
		<tr>
			<td>Trypan blue stain</td>
			<td>Life Technologies</td>
			<td>T10282</td>
			<td>Cell counting</td>
		</tr>
	</tbody>
</table>

technical applications of microelectrode array and patch clamp recordings on human induced pluripotent stem cell-derived cardiomyocytes

Drug-induced cardiotoxicity is the leading cause of drug attrition and withdrawal from the market. Therefore, using appropriate preclinical cardiac safety assessment models is a critical step during drug development. Currently, cardiac safety assessment is still highly dependent on animal studies. However, animal models are plagued by poor translational specificity to humans due to species-specific differences, particularly in terms of cardiac electrophysiological characteristics. Thus, there is an urgent need to develop a reliable, efficient, and human-based model for preclinical cardiac safety assessment. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as an invaluable in vitro model for drug-induced cardiotoxicity screening and disease modeling. hiPSC-CMs can be obtained from individuals with diverse genetic backgrounds and various diseased conditions, making them an ideal surrogate to assess drug-induced cardiotoxicity individually. Therefore, methodologies to comprehensively investigate the functional characteristics of hiPSC-CMs need to be established. In this protocol, we detail various functional assays that can be assessed on hiPSC-CMs, including the measurement of contractility, field potential, action potential, and calcium handling. Overall, the incorporation of hiPSC-CMs into preclinical cardiac safety assessment has the potential to revolutionize drug development.

This protocol describes how to measure the contraction motion, field potential, action potential, and Ca2+ transient of hiPSC-CMs. A schematic diagram including the enzymatic digestion, cell seeding, maintenance, and functional assay conduction is shown in Figure 1. The formation of the hiPSC-CM monolayer is necessary for the contraction motion measurement (Figure 2B). A representative trace of the contraction-relaxation motion of hiPSC-CMs is shown i...

Watch this Scientific Journal Video about Technical Applications of Microelectrode Array and Patch Clamp Recordings on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes at JoVE.com

Technical Applications of Microelectrode Array and Patch Clamp Recordings on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a promising in vitro model for drug-induced cardiotoxicity screening and disease modeling. Here, we detail a protocol for measuring the contractility and electrophysiology of hiPSC-CMs.

Drug-induced cardiotoxicity is the leading cause of drug attrition and withdrawal from the market. Therefore, using appropriate preclinical cardiac safety ...

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2.7K Views.  Stanford University School of Medicine. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a promising in vitro model for drug-induced cardiotoxicity screening and disease modeling. Here, we detail a protocol for measuring the contractility and electrophysiology of hiPSC-CMs.

Video: Technical Applications of Microelectrode Array and Patch Clamp Recordings on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs&#160;that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d&#160;with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming.

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Here we report a rapid and efficient gene editing method based on RMCE in the AAVS1 locus of human Pluripotent Stem Cells (hPSCs) that improves upon previously described systems. Using this technique, isogenic lines can be rapidly and reliably generated for proper comparative studies, facilitating transgenesis-mediated research with hPSCs.

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is ...

Targeted and Selective Treatment of Pluripotent Stem Cell-derived Teratomas Using External Beam Radiation in a Small-animal Model

The growing number of victims of &#34;stem cell tourism,&#34; the unregulated transplantation of stem cells worldwide, has raised concerns about the safety of stem cell transplantation. Although the transplantation of differentiated rather than undifferentiated cells is common practice, teratomas can still arise from the presence of residual undifferentiated stem cells at the time of transplant or from spontaneous mutations in differentiated cells. Because stem cell therapies are often delivered into anatomically sensitive sites, even small tumors can be clinically devastating, resulting in blindness, paralysis, cognitive abnormalities, and cardiovascular dysfunction. Surgical access to these sites may also be limited, leaving patients with few therapeutic options. Controlling stem cell misbehavior is, therefore, critical for the clinical translation of stem cell therapy.
External beam radiation offers an effective means of delivering targeted therapy to decrease the teratoma burden while minimizing injury to surrounding organs. Additionally, this method avoids genetic manipulation or viral transduction of stem cells-which are associated with additional clinical safety and efficacy concerns. Here, we describe a protocol to create pluripotent stem cell-derived teratomas in mice and to apply external beam radiation therapy to selectively ablate these tumors in vivo.

Research on treatment strategies for pluripotent stem cell-derived teratomas is important for the clinical translation of stem cell therapy. Here, we describe a protocol to, first, generate stem cell-derived teratomas in mice and, then, to selectively target and treat these tumors in vivo using a small-animal irradiator.

The growing number of victims of &#34;stem cell tourism,&#34; the unregulated transplantation of stem cells worldwide, has raised concerns about the ...

Sarcomere Shortening of Pluripotent Stem Cell-Derived Cardiomyocytes using Fluorescent-Tagged Sarcomere Proteins.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells provide promising sources for cardiac disease modeling. For cardiomyopathies, sarcomere shortening is one of the standard physiological assessments that are used with adult cardiomyocytes to examine their disease phenotypes. However, the available methods are not appropriate to assess the contractility of PSC-CMs, as these cells have underdeveloped sarcomeres that are invisible under phase-contrast microscopy. To address this issue and to perform sarcomere shortening with PSC-CMs, fluorescent-tagged sarcomere proteins and fluorescent live-imaging were used. Thin Z-lines and an M-line reside at both ends and the center of a sarcomere, respectively. Z-line proteins &#8212;&#160;&#945;-Actinin (ACTN2), Telethonin (TCAP), and actin-associated LIM protein (PDLIM3) &#8212; and one M-line protein &#8212; Myomesin-2 (Myom2) &#8212;&#160;were tagged with fluorescent proteins. These tagged proteins can be expressed from endogenous alleles as knock-ins or from adeno-associated viruses (AAVs). Here, we introduce the methods to differentiate mouse and human pluripotent stem cells to cardiomyocytes, to produce AAVs, and to perform and analyze live-imaging. We also describe the methods for producing polydimethylsiloxane (PDMS) stamps for a patterned culture of PSC-CMs, which facilitates the analysis of sarcomere shortening with fluorescent-tagged proteins. To assess sarcomere shortening, time-lapse images of the beating cells were recorded at a high framerate (50-100 frames per second) under electrical stimulation (0.5-1 Hz). To analyze sarcomere length over the course of cell contraction, the recorded time-lapse images were subjected to SarcOptiM, a plug-in for ImageJ/Fiji. Our strategy provides a simple platform for investigating cardiac disease phenotypes in PSC-CMs.

This method can be used to examine sarcomere shortening using pluripotent stem cell-derived cardiomyocytes with fluorescent-tagged sarcomere proteins.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells ...

Directed Induction of Retinal Organoids from Human Pluripotent Stem Cells

Retinal cell transplantation is a promising therapeutic approach, which could restore the retinal architecture and stabilize or even improve the visual capabilities to the degenerated retina. Nevertheless, progress in cell replacement therapy presently faces the challenges of requiring an off-the-shelf source of high quality and standardized human retinas. Therefore, an easy and stable protocol is needed for the experiments. Here, we develop an optimized protocol, based on a self-organizing method with the use of exogenous molecules and reagent A as well as manual excision to generate the three-dimensional human retina organoids (RO). The human Pluripotent Stem Cells (PSCs)-derived RO expresses specific markers for photoreceptors. With the addition of COCO, a multifunctional antagonist, the differentiation efficiency of photoreceptor precursors and cones is significantly increased. The efficient use of this system, which has the benefits of cell lines and primary cells, and without the sourcing issues associated with the latter, could produce confluent retinal cells, especially photoreceptors. Thus, the differentiation of PSCs to RO provides an optimal and biorelevant platform for disease modelling, drug screening and cell transplantation.

Using a self-organizing method, we develop a protocol with the addition of COCO that could significantly increase the generation of photoreceptors.

Retinal cell transplantation is a promising therapeutic approach, which could restore the retinal architecture and stabilize or even improve the visual ...

Microelectrode Array Recording of Sinoatrial Node Firing Rate to Identify Intrinsic Cardiac Pacemaking Defects in Mice

The sinoatrial node (SAN), located in the right atrium, contains the pacemaker cells of the heart, and dysfunction of this region can cause tachycardia or bradycardia. Reliable identification of cardiac pacemaking defects requires the measurement of intrinsic heart rates by largely preventing the influence of the autonomic nervous system, which can mask rate deficits. Traditional methods to analyze intrinsic cardiac pacemaker function include drug-induced autonomic blockade to measure in vivo heart rates, isolated heart recordings to measure intrinsic heart rates, and sinoatrial strip or single-cell patch-clamp recordings of sinoatrial pacemaker cells to measure spontaneous action potential firing rates. However, these more traditional techniques can be technically challenging and difficult to perform. Here, we present a new methodology to measure intrinsic cardiac firing rate by performing microelectrode array (MEA) recordings of whole-mount sinoatrial node preparations from mice. MEAs are composed of multiple microelectrodes arranged in a grid-like pattern for recording in vitro extracellular field potentials. The method described herein has the combined advantage of being relatively faster, simpler, and more precise than previous approaches for recording intrinsic heart rates, while also allowing easy pharmacological interrogation.

This protocol aims to describe a new methodology to measure intrinsic cardiac firing rate using microelectrode array recording of the whole sinoatrial node tissue to identify pacemaking defects in mice. Pharmacological agents can also be introduced in this method to study their effects on intrinsic pacemaking.

The sinoatrial node (SAN), located in the right atrium, contains the pacemaker cells of the heart, and dysfunction of this region can cause tachycardia or ...

Ultrasound-Guided Induced Pluripotent Stem Cell-Derived Cardiomyocyte Implantation in Myocardial Infarcted Mice

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a promising cell source for cardiac repair after ischemic damage. However, a low grafted rate is a significant obstacle for effective cardiac tissue regeneration after transplantation. This protocol shows that multiple hiPSC-CM ultrasound-guided percutaneous injections into an MI area effectively increase cell transplantation rates. The study also describes the entire hiPSC-CM culture process, pretreatment, and ultrasound-guided percutaneous delivery methods. In addition, the use of human mitochondrial DNA help detect the absence of hiPSC-CMs in other mouse organs. Lastly, this paper describes the changes in cardiac function, angiogenesis, cell size, and apoptosis at the infarcted border zone in mice 4 weeks after cell delivery. It can be concluded that echocardiography-guided percutaneous injection of the left ventricular myocardium is a feasible, relatively invasive, satisfactory, repeatable, and effective cellular therapy.

Ultrasound-guided cell delivery around the site of myocardial infarction in mice is a safe, effective, and convenient way of cell transplantation.

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem ...

Laser-Induced Action Potential-Like Measurements of Cardiomyocytes on Microelectrode Arrays for Increased Predictivity of Safety Pharmacology

Life-threatening drug-induced cardiac arrhythmia is often preceded by prolonged cardiac action potentials (AP), commonly accompanied by small proarrhythmic membrane potential fluctuations. The shape and time course of the repolarizing fraction of the AP can be pivotal for the presence or absence of arrhythmia.
Microelectrode arrays (MEA) allow easy access to cardiotoxic compound effects via extracellular field potentials (FP). Although a powerful and well-established tool in research and cardiac safety pharmacology, the FP waveform does not allow to infer the original AP shape due to the extracellular recording principle and the resulting intrinsic alternating current (AC) filtering.
A novel device, described here, can repetitively open the membrane of cardiomyocytes cultivated on top of the MEA electrodes at multiple cultivation time points, using a highly focused nanosecond laser beam. The laser poration results in transforming the electrophysiological signal from FP to intracellular-like APs (laser-induced AP, liAP) and enables the recording of transcellular voltage deflections. This intracellular access allows a better description of the AP shape and a better and more sensitive classification of proarrhythmic potentials than regular MEA recordings. This system is a revolutionary extension to the existing electrophysiological methods, permitting accurate evaluation of cardiotoxic effect with all advantages of MEA-based recordings (easy, acute, and chronic experiments, signal propagation analysis, etc.).

The combination of laser poration and microelectrode arrays (MEA) allows action potential-like recordings of cultivated primary and stem cell-derived cardiomyocytes. The waveform shape provides superior insight into test compounds' mode of action than standard recordings. It links patch-clamp and MEA readout to further optimize cardio safety research in the future.

Life-threatening drug-induced cardiac arrhythmia is often preceded by prolonged cardiac action potentials (AP), commonly accompanied by small ...

Voltage-Dependent Potassium Current Recording on H9c2 Cardiomyocytes via the Whole-Cell Patch-Clamp Technique

Potassium channels on the myocardial cell membrane play an important role in the regulation of cell electrophysiological activities. Being one of the main ion channels, voltage-gated potassium (Kv) channels are closely associated with some serious heart diseases, such as drug-induced myocardial damage and myocardial infarction. In the present study, the whole-cell patch-clamp technique was employed to determine the effects of 1.5 mM 4-aminopyridine (4-AP, a broad-spectrum potassium channel inhibitor) and aconitine (AC, 25 &#181;M, 50 &#181;M, 100 &#181;M, and 200 &#181;M) on the Kv channel current (IKv) in H9c2 cardiomyocytes. It was found that 4-AP inhibited the IKv by about 54%, while the inhibitory effect of AC on the IKv showed a dose-dependent trend (no effect for 25 &#181;M, 30% inhibitory rate for 50 &#181;M, 46% inhibitory rate for 100 &#181;M and 54% inhibitory rate for 200 &#181;M). Due to the characteristics of higher sensitivity and precision, this technique will promote the exploration of cardiotoxicity and the pharmacological effects of ethnomedicine targeting ion channels.

Voltage-Dependent Potassium Current Recording on H9c2 Cardiomyocytes via the Whole-Cell Patch-Clamp Technique

The present protocol describes an efficient method for the real-time and dynamic acquisition of voltage-gated potassium (Kv) channel currents in H9c2 cardiomyocytes using the whole-cell patch-clamp technique.

Potassium channels on the myocardial cell membrane play an important role in the regulation of cell electrophysiological activities. Being one of the main ...