Name: high-throughput cardiotoxicity screening using mature human induced pluripotent stem cell-derived cardiomyocyte monolayers
Uploaded: 2023-03-24T14:30:00
Description: Watch this Scientific Journal Video about High-Throughput Cardiotoxicity Screening Using Mature Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Monolayers at JoVE.com

This work has been supported by NIH grants HL148068-04 and R44ES027703-02 (TJH).

hiPSC usage in this protocol was approved by the University of Michigan HPSCRO Committee (Human Pluripotent Stem Cell Oversight Committee). See the Table of Materials for a list of materials and equipment. See Table 1 for media and their compositions.
1. Thawing and plating commercially available cryopreserved hiPSC-CMs for maturation on a maturation-inducing extracellular matrix (MECM)
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	<li>Warm all the reagents to room temperature and...

<ol>
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E., Anzai, T., Chanthra, N., Uosaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+brief+review+of+current+maturation+methods+for+human+induced+pluripotent+stem+cells-derived+cardiomyocytes.">A brief review of current maturation methods for human induced pluripotent stem cells-derived cardiomyocytes.</a> Frontiers in Cell and Developmental Biology. 8, 178 (2020).</li><li>Guo, Y., Pu, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyocyte+maturation:+new+phase+in+development.">Cardiomyocyte maturation: new phase in development.</a> Circulation Research. 126 (8), 1086-1106 (2020).</li><li>Yang, X., Pabon, L., Murry, C. 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There are several different approaches to in vitro cardiotoxicity screening using hiPSC-CMs. A recent &#34;Best Practices&#34; paper on the use of hiPSC-CMs presented the various in vitro assays, their primary readouts, and importantly, each assay&#39;s granularity to quantify human cardiac electrophysiological function20. In addition to using membrane-piercing electrodes, the most direct measure of human cardiac electrophysiological function is provided by VSDs. VSD assay readou...

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been validated on an international scale, and are available for in vitro cardiotoxicity screening1. Highly pure hiPSC-CMs can be generated in virtually unlimited numbers, cryopreserved, and thawed. Upon replating, they also reanimate and begin contracting with a rhythm reminiscent of the human heart2,3. Remarkably, individual hiPSC-CMs couple to each other and form functional syncytia that beat as a single tissue. Nowadays, hiPSCs are routinely derived from patient blood samples, so any person can be represented using in vitro hiPSC-CM cardiotoxicity screening assays4,5. This creates the opportunity to perform &#34;Clinical Trials in a Dish&#34;, with significant representation from diverse populations6.
One critical advantage over existing animal and animal cell cardiotoxicity screening approaches is that hiPSC-CMs utilize the full human genome and offer an in vitro system with genetic similarities to the human heart. This is especially attractive for pharmacogenomics and personalized medicine - the use of hiPSC-CMs for medication and other therapy development is predicted to provide more accurate, precise, and safe medication prescriptions. Indeed, two-dimensional (2D) hiPSC-CM monolayer assays have proven to be predictive of medication cardiotoxicity, using a panel of clinically used medications with a known risk of causing arrhythmias1,7,8,9. Despite the vast potential of hiPSC-CMs and the promise to streamline and make drug development cheaper, there has been a reluctance to use these novel assays10,11,12.
Until now, one major limitation of widespread adoption and acceptance of hiPSC-CM screening assays is their immature, fetal-like appearance, as well as their function. The critical issue of hiPSC-CM maturation has been reviewed and debated in the scientific literature ad nauseum13,14,15,16. Likewise, many approaches have been employed to promote hiPSC-CM maturation, including extracellular matrix (ECM) manipulations in 2D monolayers and the development of 3D engineered heart tissues (EHTs)17,18. At the moment, there is a widely held belief that the use of 3D EHTs will provide superior maturation relative to 2D monolayer-based approaches. However, 2D monolayers provide a higher efficiency of cell utilization and increased success in plating compared to 3D EHTs; 3D EHTs utilize greater numbers of cells, and often require the inclusion of other cell types that can confound results. Therefore, in this article, the focus is on using a simple method to mature hiPSC-CMs cultured as 2D monolayers of electrically and mechanically coupled cells.
Advanced hiPSC-CM maturation can be achieved in 2D monolayers using an ECM. The 2D monolayers of hiPSC-CMs can be matured using a soft, flexible polydimethylsiloxane coverslip, coated with basement membrane matrix secreted by an Engelbreth-Holm-Swarm mouse sarcoma cell (mouse ECM). In 2016, reports showed that hiPSC-CMs cultured on this soft ECM condition matured functionally, displaying action potential conduction velocities near adult heart values (~50 cm/s)18. Further, these mature hiPSC-CMs displayed many other electrophysiological characteristics reminiscent of the adult heart, including hyperpolarized resting membrane potential and expression of Kir2.1. More recently, reports identified a human perinatal stem cell-derived ECM coating that promotes the structural maturation of 2D hiPSC-CMs19. Here, easy-to-use methods are presented to structurally mature 2D hiPSC-CM monolayers for use in high-throughput electrophysiological screens. Further, we provide validation of an optical mapping instrument for the automated acquisition and analysis of 2D hiPSC-CM monolayer electrophysiological function, using voltage-sensitive dyes (VSDs) and calcium-sensitive probes and proteins.

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			<td>0.25% Trypsin EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
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			<td>0.5 mg/mL BSA (7.5 &#181;mol/L)</td>
			<td>Millipore Sigma</td>
			<td>A3294</td>
			<td></td>
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			<td>2.9788 g/500 mL HEPES (25 mmol/L)</td>
			<td>Millipore Sigma</td>
			<td>H4034</td>
			<td></td>
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			<td>AdGCaMP6m</td>
			<td>Vector biolabs</td>
			<td>1909</td>
			<td></td>
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			<td>Albumin human</td>
			<td>Sigma</td>
			<td>A9731-1G</td>
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			<td>alpha actinin antibody</td>
			<td>ThermoFisher</td>
			<td>MA1-22863</td>
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			<td>B27</td>
			<td>Gibco</td>
			<td>17504-044</td>
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			<td>Blebbistatin</td>
			<td>Sigma</td>
			<td>B0560</td>
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			<td>CalBryte 520AM</td>
			<td>AAT Bioquest</td>
			<td>20650</td>
			<td></td>
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			<td>CELLvo MatrixPlus 96wp</td>
			<td>StemBiosys</td>
			<td>N/A</td>
			<td>https://www.stembiosys.com/products/cellvo-matrix-plus</td>
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		<tr>
			<td>CHIR99021</td>
			<td>LC Laboratories</td>
			<td>c-6556</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear Assay medium (fluorobrite)</td>
			<td>ThermoFisher</td>
			<td>A1896701</td>
			<td>For adenovirus transduction</td>
		</tr>
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			<td>DAPI</td>
			<td>ThermoFisher</td>
			<td>62248</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM:F12</td>
			<td>Gibco</td>
			<td>11330-032</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS (Fetal Bovine Serum)</td>
			<td>Sigma</td>
			<td>F4135-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>FluoVolt</td>
			<td>ThermoFisher</td>
			<td>F10488</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Gibco</td>
			<td>14025-092</td>
			<td></td>
		</tr>
		<tr>
			<td>iCell CM maintenance media</td>
			<td>FUJIFILM/Cellular Dynamics</td>
			<td>M1003</td>
			<td></td>
		</tr>
		<tr>
			<td>iCell2 CMs</td>
			<td>FUJIFILM</td>
			<td>1434</td>
			<td></td>
		</tr>
		<tr>
			<td>Incucyte Zoom&#160;</td>
			<td>Sartorius</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>iPS DF19-9-11T.H</td>
			<td>WiCell</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoproterenol</td>
			<td>MilliporeSigma</td>
			<td>CAS-51-30-9</td>
			<td></td>
		</tr>
		<tr>
			<td>IWP4</td>
			<td>Tocris</td>
			<td>5214</td>
			<td></td>
		</tr>
		<tr>
			<td>L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate</td>
			<td>Sigma</td>
			<td>A8960-5g</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>A2916801</td>
			<td></td>
		</tr>
		<tr>
			<td>LS columns</td>
			<td>Miltenyii Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS Buffer (autoMACS Running Buffer)</td>
			<td>Miltenyii Biotec</td>
			<td>130-091-221</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354234</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoTracker Red</td>
			<td>ThermoFisher</td>
			<td>M7512</td>
			<td></td>
		</tr>
		<tr>
			<td>Nautilus HTS Optical Mapping&#160;</td>
			<td>CuriBio</td>
			<td></td>
			<td>https://www.curibio.com/products-overview</td>
		</tr>
		<tr>
			<td>Nikon A1R Confocal Microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>nonessential amino acids</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>pre-separation filter</td>
			<td>Miltenyii Biotec</td>
			<td>130-041-407</td>
			<td></td>
		</tr>
		<tr>
			<td>PSC-Derived Cardiomyocyte Isolation Kit, human</td>
			<td>Miltenyii Biotec</td>
			<td>130-110-188</td>
			<td></td>
		</tr>
		<tr>
			<td>Pulse</td>
			<td>CuriBio</td>
			<td></td>
			<td>https://www.curibio.com/products-overview</td>
		</tr>
		<tr>
			<td>Quadro MACS separator (Magnet)</td>
			<td>Miltenyii Biotec</td>
			<td>130-091-051</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Sigma</td>
			<td>R2625</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640&#160;</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 (+HEPES, +L-Glutamine)</td>
			<td>Gibco</td>
			<td>22400-089</td>
			<td></td>
		</tr>
		<tr>
			<td>StemMACS iPS-Brew XF</td>
			<td>Miltenyii Biotec</td>
			<td>130-107-086</td>
			<td></td>
		</tr>
		<tr>
			<td>TnI antibody (pan TnI)</td>
			<td>Millipore Sigma</td>
			<td>MAB1691&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Versene (ethylenediaminetetraacetic acid - EDTA solution)</td>
			<td>Gibco</td>
			<td>15040-066</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride</td>
			<td>Tocris</td>
			<td>1254</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985023</td>
			<td></td>
		</tr>
	</tbody>
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high-throughput cardiotoxicity screening using mature human induced pluripotent stem cell-derived cardiomyocyte monolayers

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical cardiotoxicity testing. In two-dimensional monolayer formats, hiPSC-CMs recapitulate the structure and function of the adult human heart muscle cells when cultured on an optimal extracellular matrix (ECM). A human perinatal stem cell-derived ECM (maturation-inducing extracellular matrix-MECM) matures the hiPSC-CM structure, function, and metabolic state in 7 days after plating. 
Mature hiPSC-CM monolayers also respond as expected to clinically relevant medications, with a known risk of causing arrhythmias and cardiotoxicity. The maturation of hiPSC-CM monolayers was an obstacle to the widespread adoption of these valuable cells for regulatory science and safety screening, until now. This article presents validated methods for the plating, maturation, and high-throughput, functional phenotyping of hiPSC-CM electrophysiological and contractile function. These methods apply to commercially available purified cardiomyocytes, as well as stem cell-derived cardiomyocytes generated in-house using highly efficient, chamber-specific differentiation protocols. 
High-throughput electrophysiological function is measured using either voltage-sensitive dyes (VSDs; emission: 488 nm), calcium-sensitive fluorophores (CSFs), or genetically encoded calcium sensors (GCaMP6). A high-throughput optical mapping device is used for optical recordings of each functional parameter, and custom dedicated software is used for electrophysiological data analysis. MECM protocols are applied for medication screening using a positive inotrope (isoprenaline) and human Ether-a-go-go-related gene (hERG) channel-specific blockers. These resources will enable other investigators to successfully utilize mature hiPSC-CMs for high-throughput, preclinical cardiotoxicity screening, cardiac medication efficacy testing, and cardiovascular research.

hiPSC-CM maturation characterized by phase contrast and immunofluorescent confocal imaging 
The timeline for ECM-mediated maturation of commercially available hiPSC-CMs using MECM coated 96-well plates is presented in Figure 1A. These data are collected using commercially available cardiomyocytes that arrive in the laboratory as cryopreserved vials of cells. Each vial contains &#62;5 &#215; 106 viable cardiomyocytes. The cells are ~98% pure and rigorously tes...

Watch this Scientific Journal Video about High-Throughput Cardiotoxicity Screening Using Mature Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Monolayers at JoVE.com

High-Throughput Cardiotoxicity Screening Using Mature Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Monolayers

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer an alternative to using animals for preclinical cardiotoxicity screening. A limitation to the widespread adoption of hiPSC-CMs in preclinical toxicity screening is the immature, fetal-like phenotype of the cells. Presented here are protocols for robust and rapid maturation of hiPSC-CMs.

Human induced stem cell-derived cardiomyocytes (hiPSC-CMs) are used to replace and reduce the dependence on animals and animal cells for preclinical ...

This protocol is significant because it enables researchers to mature commercial or in-house-made hiPSC-CMs to an adult-like phenotype that significantly improves the predictive value of hiPSC-CMs in cardiotoxicity screening. The main advantage of this protocol is that it delivers mature hiPSC-CMs in a high throughput format for optical mapping of calcium or voltage change. This protocol may be used to investigate disease mechanisms or to screen drugs for cardiotoxicity.Demonstrating the procedure will be Jeffrey Creech, a research associate from my laboratory. Begin by washing the maturation-inducing extracellular matrix, or MECM, plates two times with 200 microliters of Hank's balanced salt solution, or HBSS, 1 hour prior to cardiomyocyte plating. Keep the wells hydrated.Transfer the cardiomyocyte tubes from the liquid nitrogen tank to dry ice, and release the pressure by slightly opening the tube caps. Next, reseal the tube caps and thaw them in the water bath for 4 minutes. After the thawing of the cells, spray the tubes with 70%ethanol before opening them.Transfer the cells into 15-milliliter conical tubes with a 1-milliliter pipette. Add 1 milliliter of plating medium to the cryovial and transfer the wash into the 15-milliliter conical tube. Then, slowly drip 1 milliliter of plating medium and agitate the tube.Repeat this until the transfer of 8 milliliters of plating medium. Centrifuge the 15-milliliter tube at 300 x g for 5 minutes and resuspend the pellet in 1 milliliter of the plating medium. Remove an aliquot, and perform live cell counting with a hemocytometer before adding a plating medium to obtain 7.5 x 10 to the 5th cells per milliliter.Dispense 100 microliters of the cell suspension per well of an MECM-coated 96-well plate using a multichannel pipette, and incubate the cells for 2 days. Next, replace the medium with 200 microliters of maintenance medium, and maintain the plates for 7 days, with a change of medium on day 5. On day 7, perform EP assay, as described in the text.Ensure to change the medium every other day when opting for extending the cell culture. To purify hiPSC-CM via magnetic-activated cell sorting, or MACS, aspirate the cell culture medium and wash the cells with 1 milliliter of HBSS-Then, dissociate the cells by adding 1 milliliter of 0.25%trypsin-ethylenediaminetetraacetic, or EDTA, and incubating the cells for 10 minutes. Then, inactivate the trypsin/EDTA by resending and singularizing the cells with 2 milliliters of plating medium.From the six wells, collect the cells into a 50-milliliter conical tube using a 70-micrometer strainer. After washing the strainer with 3 milliliters of plating medium, count the cells. After counting, centrifuge and wash the cell pellet with 2 milliliters of ice-cold MACS separation buffer before pelleting and resending the cells in 80 microliters of cold MACS separation buffer per 5 x 10 to the 6th cells.Add 20 microliters of cold non-cardiomyocyte depletion cocktail and mix the suspension before incubating on ice for 10 minutes. After washing the cells with 4 milliliters of cold MACS separation buffer at 300 x g for 5 minutes, resuspend the pellet in 80 microliters of cold MACS separation buffer. Add 20 microliters of cold anti-biotin microbeads per 5 x 10 to the 6th cells, and gently mix the cell suspension before incubating on ice for 10 minutes.While the samples are incubating, place the positive depletion columns fitted with the 30 micrometer pre-separation filters on the MACS separator, and place the labeled 15-milliliter collection tubes below the columns. Next, prime each column with 3 milliliters of cold MACS separation buffer. Mix the antibody-treated cell suspension with 2 milliliters of MACS separation buffer and add it to the column.Add 2 milliliters of MACS separation buffer to each column and collect 12 milliliters of flowthrough cardiomyocyte suspension. Centrifuge the cardiomyocytes and discard the supernatant before resuspending the cardiomyocytes in a 1-milliliter plating medium. To determine the concentration, count the cells and adjust the volume to the desired seeding density before plating the purified cardiomyocyte cells on the MECM 96-well plates.Aspirate and replace the cardiomyocyte maintenance medium with 100 microliters of voltage-sensitive dyes, or VSD, or calcium-sensitive fluorophores, or CSF, per well of a 96-well plate. After 30 minutes of incubation, replace the dyes with an assay medium or HBSS. Equilibrate the cells at 37 degrees Celsius before acquiring baseline data optical mapping using a high-throughput optical mapping device.To treat the cells with drugs for acute exposure testing or map the cells chronically exposed to drugs of interest, dilute the drugs in dimethyl sulfoxide, or DMSO, and store them as stock solutions at 20 degrees Celsius before diluting them in HBSS to the desired concentrations. For cardiotoxicity testing in 96-well plates, add four doses of a compound with at least eight wells per dose. Use doses ranging from below to above the clinically-effective therapeutic plasma concentration, including the effective dose.Similar to the baseline electrophysiology measurements before drug application, capture electrophysiology recordings at least 30 minutes after drug treatment for chronic studies. After baseline recordings, apply at least four doses of each medication to mature hiPSC-CMs monolayers expressing GCaMP6m, with at least eight wells per dose. Equilibrate the medications on the cells for 30 minutes, and warm the cells to 37 degrees Celsius before and during the data acquisition.Following baseline recordings of an entire plate, add 500 nanomolar isoproterenol to every well to enable robust drug response data and quantify the effects of isoproterenol on monolayer beat rate, contraction amplitude, and calcium transient duration. To acquire optical mapping data, open the front drawer and position the plate on the plate heater. After opening the acquisition software and determining the file saving location in the software, select 10 to 30 seconds duration and frame rate of acquisition for higher temporal resolution, and click Start Acquisition.Open the analysis software, and in the Import Filter tab, select either Browse for a single file or Tile Multiple to reconstruct a plate. Select the files and enter a number of rows and columns and select Automatic. Next, select Update Pixel Size, enter distance per pixel, and use the Well Wizard to determine the wells'location in the image before clicking Process Save and moving to the next tab.Open the ROIs, or Regions of Interest tab, and choose to draw the ROIs manually, automatically, or not use ROIs at all, which will be considered for the analysis of the entire plate. After selecting the region in the well, click the Process Save button to move to the next step. Check the Hide Wells and Only Show Filtered boxes to visualize the ROIs.Open the Analysis tab, select each well or ROI to confirm the accuracy of automatic beat detection. Add or remove beats from the traces by selecting an individual beat and pressing Delete on the keyboard. Once done, click on Save.Next, click the Time-Space Plot button in the Analysis tab and add the line to determine the time-space plot to visualize data for each well or the ROI. After selecting a horizontal or vertical line, click on Generate Plot. After ensuring the accurate beat detection, proceed to the Export tab and select File Format, enter Beat Statistic option before clicking Export.Once exported, open data files and run the chosen statistical analysis routine. Phase contrast imaging showed that the hiPSC-CMs plated on the MECM matured and became structurally distinct from the same batch of hiPSC-CMs replated on the mouse ECM. Mature cells became rod-shaped, while immature cells retained a circular shape.Cardiomyocytes stained with alpha-actinin antibodies showed the typical shape of the cells cultured on each ECM condition. Consistent with the TnI staining, the alpha-actinin staining indicated that the hiPSC-CMs matured on the MECM promoted a rod-shaped mature phenotype and induced greater sarcomere organization. Mitochondrial content and activity were distinct between cells cultured on the mouse ECM and the MECM.The electrophysiological data for action potentials recorded using VSDs and the optical mapping system showed that the atrial-specific cells have a significantly faster spontaneous beat rate and shorter action potential duration, or APD80, than the ventricular-specific cells. Whole-plate heat maps for parameters such as APD80 revealed the reproducibility of a given parameter within a plate. Typical action potentials of mature hiPSC-CM monolayers indicated the action potential morphology of isolated and tested adult cardiomyocytes.A typical action potential spontaneous rhythm was also displayed. In response to isoproterenol, activation of the beta-1-adrenergic receptors caused positive chronotropy, positive inotropy, and positive lusitropy. HiPSC-CM response to human Ether-a-go-go-related gene, or hERG, channel blocker, including E-4031, domperidone, vandetanib, and sotalol, was studied using GCaMP6m calcium fluorescence to monitor rhythm.The study showed the detection of early after-depolarizations caused by the E-4031 hERG channel blockade. Work fast with the cells in suspension and avoid shear stress by pipetting cells gently. Additionally, perform media change carefully to avoid damaging the hiPS syncytia.Following this procedure, cells are amenable for protein, RNA, DNA, and lipids collection for analysis with investigators'favorite technique. The technique empowers researchers to investigate diseases and test for cardiotoxicity of new drugs using adult-like cardiomyocytes.

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