Name: model of ischemic heart disease and video-based comparison of cardiomyocyte contraction using hipsc-derived cardiomyocytes
Uploaded: 2020-05-05T15:00:00.000+00:00
Duration: 5 min 6 s
Description: Watch this Scientific Journal Video about Model of Ischemic Heart Disease and Video-Based Comparison of Cardiomyocyte Contraction Using hiPSC-Derived Cardiomyocytes at JoVE.com

This study was supported by JSPS KAKENHI, Fund for the Promotion of Joint International Research (Fostering Joint International Research), 17KK0168. The authors gratefully acknowledge Central Research Laboratory, Okayama University Medical School for the assistance of FACS.

1. hiPSC maintenance culture
<ol>
	<li>Coat a six-well culture plate with laminin (Table of Materials).
	<ol>
		<li>Dilute laminin to 0.5&#8239;&#956;g/mL in phosphate buffered saline (PBS).</li>
		<li>Add diluted laminin to a six-well plate at a volume of 2.0 mL/well.</li>
		<li>Incubate the plate at 37&#8239;&#176;C for 30&#8239;min.</li>
	</ol>
	</li>
	<li>Remove the laminin solution from the wells.</li>
	<li>Seed hiPSCs in 2&#8239;mL of iPSC maintenance medium (Table of Materials) onto the coated wells at a density of 3&#8239;&#215;&#8239;104&#8239;cells/well without drying the surface.</li>
	<li>Subculture the hiPSCs.
	<ol>
		<li>Prepare 0.5&#215; cell dissociation enzymes (Table of Materials) solution.
		<ol>
			<li>Mix 10 &#956;L of 0.5 M Ethylenediaminetetraacetic (EDTA) and 10 mL of PBS to make 0.5 mM EDTA/PBS.</li>
			<li>Dilute 10 mL of 1&#215; cell dissociation enzymes to 0.5&#215; by adding 10 mL of 0.5 mM EDTA/PBS.</li>
		</ol>
		</li>
		<li>Aspirate the spent medium from the wells.</li>
		<li>Add 2 mL/well of PBS to wash the cells, then discard the PBS.</li>
		<li>Add 800&#8239;&#956;L of the 0.5&#215; cell dissociation enzymes, and incubate the cells for 7&#8239;min at 37 &#176;C in a humidified incubator containing 5% CO2. 
		NOTE: 0.05% trypsin-EDTA can be used to dissociate cells.</li>
		<li>Gently remove the 0.5&#215; cell dissociation enzymes solution.</li>
		<li>Wash the cells with 2 mL PBS, then discard the PBS. 
		NOTE: Be gentle because the cells easily detach after adding cell dissociation enzymes solution.</li>
		<li>Add 1&#8239;mL of iPS maintenance medium (Table of Materials) containing 10&#8239;&#956;M Y-27632. 
		NOTE: Adding Y-27632 increases the survival rate of hiPSCs.</li>
		<li>Dislodge cells using a cell scraper, and collect them in a 15 mL centrifuge tube.</li>
		<li>Count the number of cells. Adjust the cell density to 1.5&#8239;&#215;&#8239;104&#8239;cells/mL by adding iPS maintenance medium containing 10 &#956;M Y-27632. 
		NOTE: Do not use centrifugation in order to prevent cell damage.</li>
		<li>Seed 2 mL of cell mixture on the laminin-coated six-well plate (final density: 3&#8239;&#215;&#8239;104&#8239;cells/well).</li>
		<li>Incubate the cells at 37 &#176;C in a humidified incubator containing 5% CO2.</li>
		<li>Replace the culture medium with iPS maintenance medium without Y-27632 on days 1, 4, 5, and 6.</li>
	</ol>
	</li>
	<li>Subculture the cells on day 7. 
	NOTE: Subculture the cells by day 7 in order to inhibit random differentiation of hiPSCs.</li>
</ol>
2. Induction of cardiac differentiation of hiPSCs
<ol>
	<li>Coat a 96-well culture plate with laminin (Table of Materials).
	<ol>
		<li>Dilute laminin to 1.675&#8239;&#956;g/mL with PBS.</li>
		<li>Add the diluted laminin solution to a 96-well plate at a volume of 0.1 mL/well.</li>
		<li>Incubate the plate at 37&#8239;&#176;C for 30&#8239;min.</li>
	</ol>
	</li>
	<li>Seed hiPSCs onto a 96-well plate at a density of 3&#8239;&#215;&#8239;104&#8239;cells/well.</li>
	<li>Proliferate hiPSCs at 37 &#176;C in a humidified incubator containing 5% CO2.
	<ol>
		<li>One day later, replace the medium with 200 &#956;L/well iPS growth medium (Table of Materials).</li>
		<li>Change the medium every day for an additional 2&#8211;3 days until the cells reach 70%&#8211;80% confluency.</li>
	</ol>
	</li>
	<li>Apply differentiation media.
	<ol>
		<li>Aspirate the spent medium and replace it slowly with 200 &#956;L/well of pre-warmed differentiation medium A (Table of Materials).</li>
		<li>Place the plate at 37 &#176;C in a humidified incubator containing 5% CO2.</li>
		<li>After 48 h, aspirate the medium and slowly replace it with 200 &#956;L/well of pre-warmed differentiation medium B (Table of Materials).</li>
		<li>Place the plate at 37 &#176;C in a humidified incubator containing 5% CO2. 
		NOTE: It is extremely important that the differentiation media are kept fresh. They will gradually lose their differentiation effect, typically within two weeks. If necessary, aliquot fresh medium and store at &#8722;20 &#176;C.</li>
	</ol>
	</li>
	<li>Apply cardiomyocyte maintenance medium.
	<ol>
		<li>After 48 h, aspirate the medium and slowly replace it with 200 &#956;L/well of pre-warmed cardiomyocyte maintenance medium (Table of Materials).</li>
		<li>Place the plate at 37 &#176;C in a humidified incubator containing 5% CO2.</li>
		<li>Replace the cardiomyocyte differentiation medium every other day up to day 30. 
		NOTE: It is important that the duration of the application of differentiation media A and B is accurately set to 48 h to ensure the precise expression sequence of genes required for differentiation.</li>
	</ol>
	</li>
</ol>
3. Exposure to ischemia
<ol>
	<li>Deprive the culture medium of nutrients and oxygen.
	<ol>
		<li>Prepare Dulbecco&#39;s modified Eagle medium (DMEM) without glucose and serum.</li>
		<li>Aspirate culture medium from the wells of the 96-well plate containing hiPS-CMs.</li>
		<li>Add the nutrient-deprived medium to the wells at a volume of 200 &#956;L/well.</li>
	</ol>
	</li>
	<li>Place the culture plate in a hypoxic incubator (Table of Materials).</li>
	<li>Infuse nitrogen gas, and maintain the internal oxygen concentration at 2% and CO2 concentration at 5% for 24 h.</li>
	<li>Proceed to desired analyses: e.g., viability assay, assessment of contractility, or assessment of cellular damage.</li>
</ol>
4. Assessment of cellular viability using MTT assay
<ol>
	<li>Use the MTT assay kit (Table of Materials) to quantitatively assess the viability of cells. 
	NOTE: Exposure to 1 mM hydrogen peroxide in DMEM for 1 h can be used to damage cells (positive control). Exposure to 0 mM hydrogen peroxide in DMEM can be used for the negative control.
	<ol>
		<li>Add 10 &#956;L of MTT reagent to the cells using a repeating pipettor.</li>
		<li>Mix gently for one minute on an orbital shaker.</li>
		<li>Incubate the cells for 3&#8211;4 h at 37 &#176;C in a 5% CO2 incubator. After incubation, the formazan produced in the cells will appear as dark crystals in the bottom of the wells.</li>
		<li>After removing the supernatant, dissolve the insoluble formazan crystals in 100&#8239;&#956;L of dimethyl sulfoxide solution (DMSO). This solution will dissolve the formazan crystals, producing a purple solution. 
		CAUTION: DMSO can irritate eyes, respiratory system, and skin. Wear suitable gloves and eye/face protection.</li>
		<li>Measure the absorbance of each sample with a microplate reader at a wavelength of 570&#8239;nm.</li>
	</ol>
	</li>
</ol>
5. Assessment of contractility of iPS-CMs
<ol>
	<li>If not installed, obtain Particle Image Velocimetry ImageJ plugin11 at https://sites.google.com/site/qingzongtseng/piv and install.</li>
	<li>Using a phase contrast microscope, record the video images of hiPS-CMs using the 4&#215;&#8239;objective lens at ~20 frames per second for ~10&#8239;s, and save as &#8220;analyze.avi&#8221;. For a comparison of contractility between before and after ischemia, confirm that the location of interest is recorded. 
	NOTE: A microscope with an automated stage is useful to target the location of interest. Movie file should be in .avi format for later analysis. If not, convert the movie to .avi.</li>
	<li>Create folder structure as indicated in Figure 1. An example of &#8220;joblist.txt&#8221; is indicated in supplementary file.</li>
	<li>Analyze discrete two-dimensional vector fields of cellular displacement.
	<ol>
		<li>Launch Fiji (ImageJ) software12, go to Plugins &#62; Macros &#62; Edit and open &#8220;vector_analysis.ijm&#8221; (Supplementary Coding File).</li>
		<li>Click Run. Analysis will be performed automatically. 
		NOTE: Displacement vectors D(x,y) are calculated for every 16&#8239;&#215;&#8239;16 pixels between the reference frame (the first frame) and all subsequent frames (frames 1 vs. 2, 1 vs. 3, 1 vs. 4, etc.). The result is calculated for every frame and saved as &#8220;vec_x.txt&#8221; (x is a frame number). A vector of maximum displacement, M(x, y), is defined for every (x, y) pair as follows: <img alt="Equation 1" src="/files/ftp_upload/61104/61104equ01.jpg" /> 
		where k represents the frame number at which |Dk (x,y)|&#8239;=&#8239;max [|D2 (x,y)|, |D3 (x,y)|, &#8230;, |Dn (x,y)|] and n denotes the last frame. The result is saved as &#8220;Max_vector.txt&#8221;. |M(x, y)| represents the maximum displacement caused by cardiomyocyte contraction at the analysis point (x, y). Contractility value C in arbitrary units is calculated as follows: <img alt="Equation 2" src="/files/ftp_upload/61104/61104equ02.jpg" /> 
		This value is saved at column 7, row 1 in the &#8220;Max_vector.txt&#8221;. C represents the sum of displacement for all (x, y). The larger the portion where the displacement due to myocardial contraction is large, the larger the value of C. The vector field of maximum displacement, M(x, y), is overlaid with phase contrast image of the first frame (&#8220;Phase_contrast.png&#8221;) and saved as &#8220;Overlaid.png&#8221;. As the magnitude of the displacement vector is calculated based on the first frame of the video, it is preferable for the hiPS-CMs to be at resting diastolic period for the first frame.</li>
	</ol>
	</li>
</ol>
6. Assessment of cellular damage using flow cytometry
<ol>
	<li>Dilute a 1 mg/mL solution of propidium iodide to 1:1,000 in PBS.</li>
	<li>Stain the detached cells with propidium iodide.
	<ol>
		<li>Aspirate the medium and place it in an appropriately sized centrifuge tube.</li>
		<li>Centrifuge the tube at 1,000 &#215; g for 5 min, and carefully remove the supernatant so as not to lose sedimented cells.</li>
		<li>Incubate the cells with the diluted propidium iodide solution at room temperature for 15 min in the dark.</li>
		<li>Centrifuge the tube at 1,000 &#215; g for 5 min, and carefully remove the supernatant so as not to lose sedimented cells.</li>
		<li>Reconstitute the cells in ~1 mL of PBS. 
		NOTE: It is important to collect detached floating cells from the medium to accurately quantify cellular damage.</li>
	</ol>
	</li>
	<li>Stain attached cells with propidium iodide.
	<ol>
		<li>Wash attached cells twice with PBS gently, then discard PBS.</li>
		<li>Incubate the cells with the diluted propidium iodide solution for 15 min in the dark.</li>
		<li>Aspirate the propidium iodide solution.</li>
		<li>Detach the cells using 0.25% trypsin. Move the cell solution into a centrifuge tube.</li>
		<li>Centrifuge the tube at 1,000 &#215; g for 5 min, and carefully remove the supernatant so as not to lose sedimented cells.</li>
		<li>Reconstitute the cells in ~1 mL of PBS.</li>
	</ol>
	</li>
	<li>Mix the floating and attached cells for fluorescence activated cell sorting (FACS) analysis.</li>
	<li>Pass the cell solution through a 30&#8239;&#956;m filter. 
	NOTE: Passing the cells to a filter is very important for an accurate FACS measurement.</li>
	<li>Analyze the samples using a FACS system.</li>
</ol>
7. Immunostaining
<ol>
	<li>Fix the cell sample.
	<ol>
		<li>Aspirate the culture medium.</li>
		<li>Add 4% paraformaldehyde in PBS for 10&#8239;min&#8239;at room temperature.</li>
		<li>Wash the cells three times with PBS. 
		NOTE: Fresh paraformaldehyde solution is recommended for optimal fixation.</li>
	</ol>
	</li>
	<li>Permeabilize the cells with 0.2% Triton X-100 for 15&#8239;min, then discard the reagent.</li>
	<li>Add 3% bovine serum albumin to block the cells for 30&#8239;min.</li>
	<li>Apply primary antibody.
	<ol>
		<li>Discard bovine serum albumin solution from the culture plate.</li>
		<li>Incubate the cells with primary antibodies overnight at 4&#8239;&#176;C.</li>
		<li>Remove the antibody solution.</li>
		<li>Wash the cells three times with PBS.</li>
	</ol>
	</li>
	<li>Apply secondary antibody.
	<ol>
		<li>Discard PBS solution from the culture plate.</li>
		<li>Incubate the cells with secondary antibodies for 30 min at room temperature in the dark. 
		NOTE: Primary anti-cardiac troponin T (TNNT2) mouse monoclonal antibody is used at a dilution of 1:750 in 3% BSA. The Alexa Fluor 488-conjugated secondary goat anti-mouse antibody is diluted to 1:1,000 in 3% BSA.</li>
	</ol>
	</li>
	<li>Additional staining of nucleus and actin fibers.
	<ol>
		<li>Remove the antibody solution.</li>
		<li>Incubate the cells in nucleus staining reagent (Table of Materials) and actin staining reagent (Table of Materials) in PBS for 30&#8239;min at room temperature in the dark. 
		NOTE: 4&#8217;, 6-diamidino-2-phenylindole (DAPI) or Hoechst 33342 can be used for nuclear staining.</li>
		<li>Wash the cells three times with PBS.</li>
	</ol>
	</li>
	<li>Capture fluorescence images using a fluorescence microscope.</li>
</ol>

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+oxygen+requirements+in+ischemic+cardiomyocytes.">Modeling oxygen requirements in ischemic cardiomyocytes.</a> Journal of Biological Chemistry. 292 (28), 11760-11776 (2017).</li><li>Rounds, S. A., Wilde, F. D., Ritz, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+A6.+Section+6.2.">Chapter A6. Section 6.2.</a> Dissolved oxygen. Report No. 09-A6.2. , (2006).</li><li>Al-Ani, 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=Oxygenation+in+cell+culture:+Critical+parameters+for+reproducibility+are+routinely+not+reported.">Oxygenation in cell culture: Critical parameters for reproducibility are routinely not reported.</a> PLoS One. 13 (10), 0204269 (2018).</li><li>Cadet, J. S., Kamp, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Recipe+for+T-Tubules+in+Human+iPS+Cell-Derived+Cardiomyocytes.">A Recipe for T-Tubules in Human iPS Cell-Derived Cardiomyocytes.</a> Circulation Research. 121 (12), 1294-1295 (2017).</li><li>Halloin, C., 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=Continuous+WNT+Control+Enables+Advanced+hPSC+Cardiac+Processing+and+Prognostic+Surface+Marker+Identification+in+Chemically+Defined+Suspension+Culture.">Continuous WNT Control Enables Advanced hPSC Cardiac Processing and Prognostic Surface Marker Identification in Chemically Defined Suspension Culture.</a> Stem Cell Reports. 13 (2), 366-379 (2019).</li></ol>

The authors have nothing to disclose.

Researchers often use laboratory small-animal models to conduct IHD experiments. Here, we developed a cell culture model of human IHD to conduct such experiments.
The main problem a user of this protocol may face is low rate of differentiated cardiomyocytes. Several steps should be taken with great care to improve the rate of successful differentiation: (a) be gentle when pipetting because the cells easily detach after addition of the cell dissociation enzymes reagent, (b) adding Y-27632 increases the survival rate of iPS cells when detaching, and (c) the timing of medium changes at the beginning of differentiation should be precisely 48 h apart to ensure controlled expression of genes involved in differentiation.
Regarding the extracellular matrix proteins used for coating the surface of culture plate, other materials than laminin we used in this protocol can be used. For example, Matrigel14,15, and gelatin16,17 are used for the feeder-free maintenance culture of hiPSCs. According to Haraguchi et al., hiPSCs seeded on Matrigel were successfully differentiated into cardiac cell sheet18.
There are a number of preceding studies regarding culture models for disease modeling using cardiomyocytes derived from hiPSCs. Regarding the modeling of ischemia-reperfusion injury, manipulations of the cellular environment, such as hyperkalemia, acidosis, and lactate accumulation, have been introduced19. Other methods include cell pelleting to hinder oxygen diffusion20 and metabolic inhibition using cyanide21. In the current protocol, cell injury was achieved by relatively simple method, namely 24 h deprivation of oxygen and nutrients. However, care should be taken to consider accurate pathophysiological processes of the ischemic heart disease, as there are indeed differences between the disease in vivo and the disease model of the current protocol, such as the presence or absence of three-dimensional cellular environment and blood.
Regarding the technological aspect of the evaluation of cardiomyocyte function, Toepfer et al. reported a MatLab-based algorithm to determine sarcomere contraction and relaxation in hiPS-CMs22. Smith et al. reported an advanced method of high-throughput electrophysiological analysis of excitable cells derived from hiPS-CMs, using sophisticated nanotopographically patterned multielectrode arrays23,24. The advantage of our protocol is that it only requires conventional software and consumables, such as imageJ software and 96-well plates.
With respect to the oxygen level in the heart, the oxygen pressure in veins that reach the heart is thought to be 40 mmHg25. According to McDougal et al., the extracellular oxygen pressure under hypoxia is estimated to be &#60;12.8 mmHg26. By applying the method of Rounds et al.27, the oxygen pressure in the culture medium (salinity: 35&#8240;) under the hypoxic condition (2% oxygen) at 37 &#176;C treated with the current protocol is calculated to be 14.9 mmHg, which is higher than the estimation above. Interestingly, Al-Ani et al. reported that there is a gradient of oxygen pressure in the culture medium, and the oxygen pressure is affected by cell type, seeding density, and medium volume28. Typically, the oxygen concentration at the bottom of the culture plate where cells reside is the lowest. Therefore, the effect of depth in the culture medium would further reduce the effective oxygen pressure near hiPS-CMs. In order to induce sufficient damage to hiPS-CMs using hypoxic condition, careful attention must be paid to the depth of medium and the density of cells.
Our hiPS-CM model, which is very close to the physiological conditions of human cardiac myocytes, advantageously mimics human IHD. Animal model-based approaches include ethical, technical, and academic issues. Particularly, in vivo models require an advanced technique of microsurgery to achieve reproducible data: e.g., occlusion of the anterior descending branch of the left coronary artery in rodents3. The hiPS-CM model described herein overcomes these critical barriers and provides a useful, relevant, and repeatable platform for cardiovascular disease.
However, some limitations should be noted. An obvious difference between iPS-induced cardiomyocytes and normal cardiomyocytes is the absence of T-tubules29, and we did not include humoral factors such as tissue damage induced by leukocytes and activation of a complement system in our study. Moreover, the rate of differentiated cardiomyocytes in this model (20.7 &#177; 9.6%, Supplementary Figure 3) should be improved. The recent publication by Halloin et al. describes a scalable, chemically defined method to induce hiPS-CM purity of &#62;95% by chemical WNT pathway modulators without requirement of any selection by markers30. Nevertheless, our model of human IHD is relatively simple and clinically applicable (e.g., drug screening using patient-derived iPS cells). Our model is also a unique platform to further elucidate mechanisms underlying IHDs.

Ischemic heart disease (IHD) is recognized worldwide as the leading cause of death, and it was estimated to be responsible for more than nine million fatalities in 20161. The prevalence of cardiovascular disease continues to rise, and globalization seems to have contributed to the prevalence of heart disease risk factors in developing countries. Therefore, the study of IHD is becoming increasingly more urgent2.
Experimental models of cardiovascular disease are critical for studying the mechanisms of disease, accuracy of diagnosis, and development of new therapies. Several experimental models have been proposed by many laboratories. It is of utmost importance to choose a model with significant advantages; feasibility, repeatability and similarity to human disease are key factors in the selection of cardiovascular disease models3. Human induced pluripotent stem cells (hiPSCs) carrying speci&#64257;c cardiomyopathy-associated mutations are a promising alternative to animal models4. Although many strategies have been described for inducing cardiomyocytes using iPSCs5,6,7,8,9, here we provide a simple method to produce an IHD model using cardiomyocytes differentiated from hiPSCs, in which laborious selection of cardiomyocyte using markers is not applied. In this method, cardiomyocytes are selected functionally by analyzing spontaneous contraction.
Induction of cardiomyocytes using hiPSCs avoids animal sacrifice and technically difficult surgery. Establishing animal models of IHD requires challenging surgical techniques10. Perfectly simulating the various pathophysiological aspects of human heart disease such as heart rate and action potentials from the physiology of rodents is nearly impossible. Coupled with the morality and ethics of using animal models, the development of new experimental models other than animal models is imperative. Cardiomyocytes differentiated from human iPS cells better mimic the physiological state of the human heart. In this protocol, we establish a model of IHD using cardiomyocytes derived from hiPS cells (hiPS-CMs). In our model, the deprivation of oxygen and glucose leads to a decrease in the contractile force and viability of hiPS-CMs. Our method provides a new approach to model IHD and demonstrates a new platform for the study of this disease.

<table><tbody><tr><td>Actin staining reagent (ActinRed 555 ReadyProbes Reagent)</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>R37112</td><td></td></tr><tr><td>Anti cardiac troponin T antibody</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>MA5-12960</td><td></td></tr><tr><td>Cardiomyocyte maintenance medium (PSC Cardiomyocyte Differentiation Kit)</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>A2921201</td><td></td></tr><tr><td>Cell dissociation enzymes (TrypLE Select)</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>12563011</td><td></td></tr><tr><td>Differentiation medium A (PSC Cardiomyocyte Differentiation Kit)</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>A2921201</td><td></td></tr><tr><td>Differentiation medium B (PSC Cardiomyocyte Differentiation Kit)</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>A2921201</td><td></td></tr><tr><td>FACS Aria III system</td><td>BD Biosciences, San Jose, CA, USA</td><td></td><td></td></tr><tr><td>Fluorescenct microscope</td><td>KEYENCE, Osaka, Japan</td><td>BZ-X710</td><td></td></tr><tr><td>Goat anti-mouse Alexa Fluor 488 secondary antibody</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>A28175</td><td></td></tr><tr><td>Human iPS cells</td><td>RIKEN, Tsukuba, Japan</td><td>201B7</td><td></td></tr><tr><td>Hypoxic incubator</td><td>SANYO, Osaka, Japan</td><td>MCO-175M</td><td></td></tr><tr><td>iPSC growth medium (Essential 8 Medium)</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>A1517001</td><td></td></tr><tr><td>iPSC maintenance medium (StemFit AK02N)</td><td>Ajinomoto, Tokyo, Japan</td><td>RCAK02N</td><td></td></tr><tr><td>Laminin (iMatrix-511)</td><td>Nippi, Tokyo, Japan</td><td>iMatrix-511</td><td></td></tr><tr><td>MTT assay kit (MTT Cell Proliferation Assay Kit)</td><td>Cayman Chemical Company, Ann Arbor, MI, USA</td><td>10009365</td><td></td></tr><tr><td>Nucleus staining reagent (NucBlue Fixed Cell ReadyProbes Reagent)</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>R37606</td><td></td></tr><tr><td>Triton X-100</td><td>Nacalai Tesque, Kyoto, Japan</td><td>35501-02</td><td></td></tr></tbody></table>

model of ischemic heart disease and video-based comparison of cardiomyocyte contraction using hipsc-derived cardiomyocytes

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with small-animal models such as rodents. However, the physiology of the human heart differs significantly from that of the rodent heart, underscoring the need for clinically relevant models to study heart disease. Here, we present a protocol to model ischemic heart disease using cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS-CMs) and to quantify the damage and functional impairment of the ischemic cardiomyocytes. Exposure to 2% oxygen without glucose and serum increases the percentage of injured cells, which is indicated by staining of the nucleus with propidium iodide, and decreases cellular viability. These conditions also decrease the contractility of hiPS-CMs as confirmed by displacement vector field analysis of microscopic video images. This protocol may furthermore provide a convenient method for personalized drug screening by facilitating the use of hiPS cells from individual patients. Therefore, this model of ischemic heart disease, based on iPS-CMs of human origin, can provide a useful platform for drug screening and further research on ischemic heart disease.

Successfully differentiated cells show spontaneous contraction under the microscope (Video 1). Typically, 50% of wells show spontaneous contraction in &#60;20 days (Supplementary Figure 1). Cardiac marker protein (e.g., cTnT) can be used to confirm successful differentiation (Figure 2).
Typically, cells from the ischemic group show lower viability in MTT assays (Figure 3A) and contractility (Figure 3E,F, Supplementary Figure 2) than those from normoxic control group. Also, the ratio of propidium iodide-positive cells is higher in the ischemic group than in the control group (Figure 3B&#8211;D), which indicates higher cellular damage.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61104/61104fig01.jpg" /> 
Figure 1: Folder structure for displacement vector analysis using imageJ. 
&#8220;joblist.txt&#8221; describes path to movie files each line. If there are three files to analyze, there will be three folders (movie1, movie2, and movie3), in which &#8220;analyze.avi&#8221; (the movie of interest) should be placed. After the analysis is performed by &#8220;vector_analysis.ijm&#8221; code, files will be generated (indicated in blue) in each movie folder. Information stored in each file is explained in detail in the main text. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61104/61104fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61104/61104fig02.jpg" /> 
Figure 2: Representative image of cardiac marker staining. 
Expression of the cardiac marker protein cardiac troponin T (cTnT). Blue: 4&#8217;,6-diamidino-2-phenylindole (DAPI), red: actin, green: cTnT. Inset: striated expression of cTnT, which corresponds to the sarcomere structure. Scale bar, 50 &#956;m. This figure has been modified from Wei et al.13. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61104/61104fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61104/61104fig03.jpg" /> 
Figure 3: Representative images of cellular viability assay, assessment of cellular damage, and contractility. 
(A) Comparison of cellular viability (MTT assay). Higher absorbance indicates higher viability. n = 5 for each condition. (B, C, and D) Flow cytometry analysis of propidium iodide (PI)-stained cells. Ischemic cells exhibit a reduced forward scatter intensity and increased PI fluorescence, compared with the control. (D) Percentage of PI-stained cells in the flow cytometry analysis. n = 3 for each condition. (E) Analysis of iPS-CMs contractility using ImageJ software. The red and blue vectors indicate the largest and smallest contractions, respectively. Scale bar, 100 &#956;m. (F) Quantitative analysis of the iPS-CM contractility using code. n = 3 for control and n = 8 for ischemic condition. Error bars represent standard error of the mean. For the statistical analysis, unpaired two-tailed Student&#8217;s t-test was performed. *: p &#60; 0.05, **: p &#60; 0.01. ***: p &#60; 0.0001. This figure is cited from Wei et al.13 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61104/61104fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1: Differentiated cells show spontaneous contraction under the microscope. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61104/61104_before.wmv" target="_blank">Please click here to download this video.</a>
Supplementary Figure 1: Kaplan-Meier analysis of percentage of samples without spontaneous contraction. 50% of iPS-CM samples showed contraction on day 20. On day 30, 64.4% of the samples showed contraction. n = 83. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61104/61104supfig01.jpg" target="_blank">Please click here to download this figure.</a>
Supplementary Figure 2: Assessment of cardiac contractility. Scaled contractility obtained by dividing contractility C by the number of analysis points (x, y) was plotted for control and ischemic groups before and after the exposure to 24 h hypoxia. n = 3 for control and n = 8 for ischemic condition. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61104/61104supfig02.jpg" target="_blank">Please click here to download this figure.</a>
Supplementary Figure 3: Assessment of the content of cardiomyocytes. Immunostaining of cardiac marker protein on a 96 well plate. Green: cTnT, Blue: DAPI. Rate of the differentiated cells was calculated to be 20.7 &#177; 9.6% by dividing the area of cTnT-stained region by that of DAPI-stained region. Scale bar = 1 mm. n = 4 for control condition. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61104/61104supfig03.jpg" target="_blank">Please click here to download this figure.</a>
Supplementary Coding File.&#160; <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61104/Supplementary_Coding_File.zip" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Model of Ischemic Heart Disease and Video-Based Comparison of Cardiomyocyte Contraction Using hiPSC-Derived Cardiomyocytes at JoVE.com

Model of Ischemic Heart Disease and Video-Based Comparison of Cardiomyocyte Contraction Using hiPSC-Derived Cardiomyocytes

We present a model of ischemic heart disease using cardiomyocytes derived from human induced pluripotent stem cells, together with a method for quantitative evaluation of tissue damage caused by ischemia. This model can provide a useful platform for drug screening and further research on ischemic heart disease.

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with ...

model-ischemic-heart-disease-video-based-comparison-cardiomyocyte

Nanyang Technological University

Research

JoVE Journal

Medicine

12.9K Views.  Okayama University. Hart-en vaatziekten onderzoek wordt vaak uitgevoerd bij kleine en zoogdieren zoals knaagdieren. Echter, de fysiologie van het menselijk hart verschilt aanzienlijk van die van het knaagdierhart. Ons protocol maakt gebruik van cardiomyocyten onderscheiden van menselijke iPS cellen om ischemische hart-en vaatziekten model en om de schade en functionele aantasting van ischemische cardiomyocyten te kwantificeren.Dit protocol kan een handige methode bieden voor gepersonaliseerde screening va...

Video: Model of Ischemic Heart Disease and Video-Based Comparison of Cardiomyocyte Contraction Using hiPSC-Derived Cardiomyocytes

Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for differentiating hiPSCs into cardiomyocytes (hiPSC-CMs), endothelial cells (hiPSC-ECs), and smooth muscle cells (SMCs) are often limited by low yield, purity, and/or poor phenotypic stability. Here, we present novel protocols for generating hiPSC-CMs, -ECs, and -SMCs that are substantially more efficient than conventional methods, as well as a method for combining cell injection with a cytokine-containing patch created over the site of administration. The patch improves both the retention of the injected cells, by sealing the needle track to prevent the cells from being squeezed out of the myocardium, and cell survival, by releasing insulin-like growth factor (IGF) over an extended period. In a swine model of myocardial ischemia-reperfusion injury, the rate of engraftment was more than two-fold greater when the cells were administered with the cytokine-containing patch comparing to the cells without patch, and treatment with both the cells and the patch, but not with the cells alone, was associated with significant improvements in cardiac function and infarct size.

We present three novel and more efficient protocols for differentiating human induced pluripotent stem cells into cardiomyocytes, endothelial cells, and smooth muscle cells and a delivery method that improves the engraftment of transplanted cells by combining cell injection with patch-mediated cytokine delivery.

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for ...

Developmental Biology

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

Bioengineering

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.

This protocol describes the procedure for sectioning and culturing human cardiac slices for preclinical drug testing and details the use of optical mapping for recording transmembrane voltage and intracellular calcium signals simultaneously from these slices.

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...

Hybrid Cell Analysis System to Assess Structural and Contractile Changes of Human iPSC-Derived Cardiomyocytes for Preclinical Cardiac Risk Evaluation

Cardiac contractility assessment is of immense importance for the development of new therapeutics and their safe transition into clinical stages. While human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold promise to serve as a human-relevant model in preclinical phases of drug discovery and safety pharmacology, their maturity is still controversial in the scientific community and under constant development. We present a hybrid contractility and impedance/extracellular field potential (EFP) technology, adding significant pro-maturation features to an industry-standard 96-well platform.
The impedance/EFP system monitors cellular functionality in real-time. Besides the beat rate of contractile cells, the electrical impedance spectroscopy readouts detect compound-induced morphological changes like cell density and integrity of the cellular monolayer. In the other component of the hybrid cell analysis system, the cells are cultured on bio-compliant membranes that mimic the mechanical environment of real heart tissue. This physiological environment supports the maturation of hiPSC-CMs in vitro, leading to more adult-like contractile responses including positive inotropic effects after treatment with isoproterenol, S-Bay K8644, or omecamtiv mecarbil. Parameters such as the amplitude of contraction force (mN/mm2) and beat duration also reveal downstream effects of compounds with influence on electrophysiological properties and calcium handling.
The hybrid system provides the ideal tool for holistic cell analysis, allowing preclinical cardiac risk assessment beyond the current perspectives of human-relevant cell-based assays.

The analysis of changes in contractile function and cellular integrity of human iPSC-derived cardiomyocytes is of immense importance for nonclinical drug development. A hybrid 96-well cell analysis system addresses both parameters in a real-time and physiological manner for reliable, human-relevant results, necessary for a safe transition into clinical stages.

Cardiac contractility assessment is of immense importance for the development of new therapeutics and their safe transition into clinical stages. While ...

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used in regulatory submissions. Here, we extend their use to cardiac medical device safety or performance assessments. We developed a novel method to evaluate cardiac medical device contractile properties in robustly contracting 2D hiPSC-CMs monolayers plated on a flexible extracellular matrix (ECM)-based hydrogel substrate. This tool enables the quantification of the effects of cardiac electrophysiology device signals on human cardiac function (e.g., contractile properties) with standard laboratory equipment. The 2D hiPSC-CM monolayers were cultured for 2-4 days on a flexible hydrogel substrate in a 48-well format.
The hiPSC-CMs were exposed to standard cardiac contractility modulation (CCM) medical device electrical signals and compared to control (i.e., pacing only) hiPSC-CMs. The baseline contractile properties of the 2D hiPSC-CMs were quantified by video-based detection analysis based on pixel displacement. The CCM-stimulated 2D hiPSC-CMs plated on the flexible hydrogel substrate displayed significantly enhanced contractile properties relative to baseline (i.e., before CCM stimulation), including an increased peak contraction amplitude and accelerated contraction and relaxation kinetics. Furthermore, the utilization of the flexible hydrogel substrate enables the multiplexing of the video-based cardiac-excitation contraction coupling readouts (i.e., electrophysiology, calcium handling, and contraction) in healthy and diseased hiPSC-CMs. The accurate detection and quantification of the effects of cardiac electrophysiological signals on human cardiac contraction is vital for cardiac medical device development, optimization, and de-risking. This method enables the robust visualization and quantification of the contractile properties of the cardiac syncytium, which should be valuable for nonclinical cardiac medical device safety or effectiveness testing. This paper describes, in detail, the methodology to generate 2D hiPSC-CM hydrogel substrate monolayers.

Here, we demonstrate a non-invasive cardiac medical device contractility evaluation method using 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, plated on a flexible substrate, coupled with video-based microscopy. This tool will be useful for the in vitro evaluation of the contractile properties of cardiac electrophysiology devices.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used ...

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

Presented here is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human-induced pluripotent stem cell-derived cardiomyocytes cultured in a high-throughput, multidimensional format. This three-dimensional model functions as a robust platform for disease modeling, high-throughput screenings, and maintains its functionality after cryopreservation.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We ...

Contractions of Human-iPSC-derived Cardiomyocyte Syncytia Measured with a Ca-sensitive Fluorescent Dye in Temperature-controlled 384-well Plates

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac physiology and pharmacology. Various methods have been proposed to record this spontaneous activity and to evaluate drug effects, but many of these methods suffer from limited throughput and/or physiological relevance. We developed a high-throughput screening system to quantify the effects of exogenous compounds on hiPSC-CM's beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader. We describe how to prepare the cell plates and the compound plates and how to run the automated assay to achieve high sensitivity and reproducibility. We also describe how to transform and analyze the fluorescence data to provide reliable measures of drug effects on spontaneous rhythm. This assay can be used in drug discovery programs to guide chemical optimization away from, or toward, compounds affecting human cardiac function.

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells are useful models of human cardiac physiology and pharmacology. Here we present a high-throughput screening system to quantify the effects of exogenous compounds on beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader.

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac ...

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 ...

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.

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 ...

Analyzing the &alpha;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. Despite the development of multiple differentiation protocols, the generation of iPSC cardiomyocytes resembling an adult-like phenotype remains challenging. One major aspect of cardiomyocytes maturation involves the formation of a well-organized sarcomere network to ensure high contraction capacity. Here, we present a super resolution-based approach for semi-quantitative analysis of the &#945;-actinin network in cardiomyocytes. Using photoactivated localization microscopy a comparison of sarcomere length and z-disc thickness of iPSC-derived cardiomyocytes and cardiac cells isolated from neonatal tissue was performed. At the same time, we demonstrate the importance of proper imaging conditions to obtain reliable data. Our results show that this method is suitable to quantitatively monitor the structural maturity of cardiac cells with high spatial resolution, enabling the detection of even subtle changes of sarcomere organization.

Analyzing the &#945;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The formation of a proper sarcomere network is important for the maturation of iPSC-derived cardiomyocytes. We present a super resolution-based approach that allows for the quantitative evaluation of the structural maturation of stem cell derived cardiomyocytes, to improve culture conditions promoting cardiac development.

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. ...

Model of Ischemic Heart Disease and Video-Based Comparison of Cardiomyocyte Contraction Using hiPSC-Derived Cardiomyocytes

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