Name: single-cell optical action potential measurement in human induced pluripotent stem cell-derived cardiomyocytes
Uploaded: 2020-12-22T13:30:00
Description: Watch this Scientific Journal Video about Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes at JoVE.com

The authors would like to acknowledge Cairn Research Ltd. for their kind financial contribution which covered production costs of this publication. In addition, we thank Ms. Ines Mueller and Ms. Stefanie Kestel for their excellent technical support.
The authors&#8217; research is supported by the German Center for Cardiovascular Research (DZHK), the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation, VO 1568/3-1, IRTG1816 RP12, SFB1002 TPA13 and under Germany&#8217;s Excellence Strategy - EXC 2067/1- 390729940) and the Else-Kr&#246;ner-Fresenius Stiftung (EKFS 2016_A20).

1. Cellular preparations
NOTE: Human iPSCs used in this protocol were derived from healthy donors and differentiated in monolayers using fully defined small molecule modulation of WNT signaling and lactate purification techniques as previously described12,13,14. iPSC-CMs were maintained every 2-3 days with a culture medium outlined below.
<ol>
	<li>Prepare a culture medium of basal medium (RPMI 1640...

<ol>
	<li>Miller, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+fluorescent+voltage+indicators+for+studying+membrane+potential.">Small molecule fluorescent voltage indicators for studying membrane potential.</a> Current Opinion in Chemical Biology. 33, 74-80 (2016).</li><li>Liang, P., 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=Drug+screening+using+a+library+of+human+induced+pluripotent+stem+cell-derived+cardiomyocytes+reveals+disease-specific+patterns+of+cardiotoxicity.">Drug screening using a library of human induced pluripotent stem cell-derived cardiomyocytes reveals disease-specific patterns of cardiotoxicity.</a> Circulation. 127 (16), 1677-1691 (2013).</li><li>Horv&#225;th, 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=Low+resting+membrane+potential+and+low+inward+rectifier+potassium+currents+are+not+inherent+features+of+hiPSC-derived+cardiomyocytes.">Low resting membrane potential and low inward rectifier potassium currents are not inherent features of hiPSC-derived cardiomyocytes.</a> Stem Cell Reports. 10 (3), 822-833 (2018).</li><li>Salama, G., Morad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Merocyanine+540+as+an+optical+probe+of+transmembrane+electrical+activity+in+the+heart.">Merocyanine 540 as an optical probe of transmembrane electrical activity in the heart.</a> Science. 191 (4226), 485-487 (1976).</li><li>Hortigon-Vinagre, 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=The+use+of+ratiometric+fluorescence+measurements+of+the+voltage+sensitive+dye+Di-4-ANEPPS+to+examine+action+potential+characteristics+and+drug+effects+on+human+induced+pluripotent+stem+cell-derived+cardiomyocytes.">The use of ratiometric fluorescence measurements of the voltage sensitive dye Di-4-ANEPPS to examine action potential characteristics and drug effects on human induced pluripotent stem cell-derived cardiomyocytes.</a> Toxicological Sciences. 154 (2), 320-331 (2016).</li><li>Blinova, 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=International+multisite+study+of+human-induced+pluripotent+stem+cell-derived+cardiomyocytes+for+drug+proarrhythmic+potential+assessment.">International multisite study of human-induced pluripotent stem cell-derived cardiomyocytes for drug proarrhythmic potential assessment.</a> Cell Reports. 24 (13), 3582-3592 (2018).</li><li>Miller, E. 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Here we describe a basic protocol to easily acquire detailed AP profiles from isolated iPSC-CMs suitable for electrophysiological modelling and cardiac drug screening. We detect regular, robust APs from our sparsely seeded iPSC-CMs which suggests both indicator functionality and methodological fidelity.
Due to the wide spectrum of commercial methodologies for iPSC reprogramming and lack of standardization for cardiac differentiation protocols, iPSC based models can show immense variability in ...

Electrophysiological modeling of cardiomyocytes and the construction of efficient platforms for cardiac drug screening is essential for the development of therapeutic strategies for a variety of arrhythmic disorders. Rapid expansion of induced pluripotent stem cell (iPSC) technology has produced promising inroads into human disease modelling and pharmacological investigation using isolated patient derived cardiomyocytes (iPSC-CM). &#8220;Gold standard&#8221; techniques for electrophysiological characterization of these cells through patch-clamp (current-clamp) can quantify action potential (AP) morphology and duration, however, this method is incredibly complex and slow, and not well suited for high throughput data acquisition1. iPSC-CMs are regularly reported to have an increased diastolic membrane potential and increased leak current when compared to adult native cardiomyocytes2. It is suggested that smaller cell size and reduced membrane capacitance observed in iPSC-CMs may produce some systematic error when using the current-clamp technique, perhaps explaining these deviations3. In order to maximize the usefulness of an iPSC-CM platform, an additional method is valuable to increase throughput and ensure data accuracy when characterizing transmembrane voltage changes at a single cell level in iPSC-CMs.
Voltage sensitive dyes (VSD) have long been a proposed method to provide faster, non-invasive and equivalent analysis of cardiac AP kinetics comparative to those of traditional techniques4. A recent study has demonstrated the suitability of ratiometric voltage sensitive probe photometry to accurately quantify the cardiac AP5. Furthermore, the ability to readily scale up optical photometry approaches lends this technique to large scale cardiotoxicity screens critical in therapeutic drug development (e.g., CiPA). Development of standardized cardiotoxicity protocols in a blinded multi-site study using microelectrode array and voltage-sensing optical techniques has demonstrated the key value of this approach6.
Many potentiometric dyes are commercially available, and ongoing synthetic development of new probes show exciting potential for streamlining their effectiveness across a variety of cardiac and neural constructs. The ideal VSD will have augmented kinetics and sensitivity, while displaying decreased capacitive load, photobleaching and cytotoxicity. The recently synthesized VF2.1Cl (FluoVolt) expresses many of these beneficial properties largely due to its novel wire-based molecular structure, shared by other members of the new VoltageFluor (VF) family7. In contrast to common electrochromic VSDs in which simple probes molecularly and electrically conjugate to the plasma membrane, this dye consists of a passively inserted, membrane-spanning synthetic wire which pairs an electron-rich donor with a modified fluorescein fluorophore (FITC). Mechanistic details are provided in Figure 1. This dye demonstrates excellent sensitivity to membrane voltage fluctuations, displaying a 27% change in emission intensity per 100 mV as opposed to ~10% seen in other common probes at comparable speeds7. In addition, wire-based PeT systems do not directly interact with the cellular electric field which produces minimal electrical interference and negligible changes in cellular capacitive load.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61890/61890fig01.jpg" /> 
Figure 1: Chemical, spectral and mechanistic parameters of VF2.1Cl dye.&#160;(A) Chemical structure of VF2.1Cl. Molecular features to note include multiple alkyl groups within the phenylene vinylene molecular wire which facilitate insertion into the plasma membrane. A negatively charged sulfonic acid group conjugated to the FITC probe ensures fluorophore stabilization on the extracellular surface and aids near perpendicular insertion relative to the electrical field of the lipid bilayer. (B) A simplified schematic of perpendicular VF2.1Cl embedding into the plasma membrane of a target cell. (C) Absorption and emission spectra of VF2.1Cl dye. Spectra is identical to that of standard FITC and GFP probes. (D) Depiction of the mechanistic mode of action of VF2.1Cl. In resting conditions (hyperpolarized), negative intracellular voltages drive free electrons towards the rostral fluorophore. Electron abundance ensures photo-induced electron transfer (PeT) is favored as a pathway out of the excited state after the optical excitation, effectively quenching fluorescence. In contrast, a depolarized membrane potential influences downward electron movement favoring fluorescence upon optical excitation. The resulting fluorescent response is linearly related to membrane voltage and can be precisely utilized to gather detailed temporal information on cellular electrophysiological kinetics. (E) Representative brightfield (upper) and fluorescence at 470 nm (lower) images of leporine cardiomyocytes loaded with VF2.1Cl. (F) Z stack of a single loaded cardiomyocyte. Arrows indicate areas of clear localization of VF2.1Cl to the cellular membrane. Images were acquired with a spinning disk confocal system consisting of a X-lightv3 spinning disk confocal head with a 50 &#181;m pinhole pattern; LDI-7 illuminator; Prime95B camera and a PlanApo Lambda 100x objective. Scale bar: 20 &#181;m. <a href="https://www.jove.com/files/ftp_upload/61890/61890fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The FITC probe conjugated to VF2.1Cl ensures that it can be used effectively under standard and GFP filter configurations, and it only requires a single channel acquisition system, both of which are common features of fluorescent imaging platforms. Analysis of dense human iPSC-CM monolayers with this dye has been recently reported8,9,10,11. Our protocol differs to these studies due to our investigation of single, isolated iPSC-CMs, unperturbed by the electrical and paracrine influences of dense syncytial monolayers, and our use of an affordable and customizable photometry system as opposed to complex confocal or wide-field imaging arrangements.
Here, we describe our protocol for the rapid acquisition and analysis of robust optical APs from isolated human iPSC-derived cardiomyocytes and native cardiomyocytes (see Supplementary File). We use VF2.1Cl coupled with a customizable state of the art platform for single cell photometry measurements.&#160;These experimental protocols have been approved by the ethics committee of the University Medical Center G&#246;ttingen (No. 10/9/15).

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	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.25 Trypsin EDTA&#160;</td>
			<td>Gibco&#160;</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement&#160;</td>
			<td>Gibco&#160;</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Carl Roth&#160;</td>
			<td>HN04.2</td>
			<td></td>
		</tr>
		<tr>
			<td>D(+)-Glucose anhydrous&#160;BioChemica</td>
			<td>ITW Reagents</td>
			<td>A1422</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum&#160;</td>
			<td>Gibco&#160;</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>FluoVolt Membrane Potential Kit&#160;</td>
			<td>Invitrogen&#160;</td>
			<td>F10488</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Carl Roth&#160;</td>
			<td>HN77.4</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>6781.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Lamanin</td>
			<td>Sigma-Aldrich</td>
			<td>114956-81-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel&#160;</td>
			<td>BD</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>9265.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Nifedipine&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>21829-25-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Invitrogen&#160;</td>
			<td>15140</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCK Inhibitor Y27632</td>
			<td>Stemolecule</td>
			<td>04-0012-10</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium&#160;</td>
			<td>Gibco&#160;</td>
			<td>61870010</td>
			<td></td>
		</tr>
		<tr>
			<td>Versene EDTA&#160;</td>
			<td>Gibco&#160;</td>
			<td>15040033</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>495LP Dichroic Beamsplitter&#160;</td>
			<td>Chroma Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Axopatch 200B Amplifier&#160;</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Circle Coverslips, Thickness 0</td>
			<td>Thermo Scientific</td>
			<td>CB00100RA020MNT0</td>
			<td></td>
		</tr>
		<tr>
			<td>Digidata 1550B</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dual OptoLED Power Supply&#160;</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ET470/40x Excitation Filter&#160;</td>
			<td>Chroma Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ET535/50m</td>
			<td>Chroma Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Etched Neubauer Hemacytometer</td>
			<td>Hausser Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Filter Cubes&#160;</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IX73 Inverted Microscope&#160;</td>
			<td>Olympus&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MonoLED</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multiport Adaptors&#160;</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Myopacer Cell Stimulator&#160;</td>
			<td>IonOptix</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optomask Shutter&#160;</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optoscan System Controller</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PH-1 Temperature Controlled Platform&#160;&#160;</td>
			<td>Warner Instruments&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Photomultiplier Detector&#160;</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PMT Amplifier Insert</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PMT Supply Insert</td>
			<td>Cairn Research&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RC-26G Open Bath Chamber&#160;</td>
			<td>Warner Instruments&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SA-OLY/2AL Stage Adaptor&#160;</td>
			<td>Olympus&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T565lpxr Dichroic Beamsplitter&#160;</td>
			<td>Chroma Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T660lpxr Dichroic Beamsplitter</td>
			<td>Chroma Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TC-20 Dual Channel Temperature Controller&#160;</td>
			<td>npi Electronic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>UPLFLN 40X Objective</td>
			<td>Olympus&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>USB 3.0 Colour Camera&#160;</td>
			<td>Imaging Source</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clampex 11.1</td>
			<td>Molecular Devices&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clampfit 11.1</td>
			<td>Molecular Devices&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IC Capture 2.4&#160;</td>
			<td>Imaging Source&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Prism 8</td>
			<td>Graphpad</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

single-cell optical action potential measurement in human induced pluripotent stem cell-derived cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61890/61890fig03.jpg" /> 
Figure 3: Optical action potential (AP) profiles of isolated native cardiomyocytes and human induced pluripotent stem cell derived cardiomyocytes (iPSC-CM).&#160;(A) Representative optical AP of a single murine cardiomyocyte (center) with Mean &#177; SEM of APD50 and APD90 (n = 7, right). (B) Representative optical AP of a singl...

Watch this Scientific Journal Video about Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes at JoVE.com

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

Research

JoVE Journal

Bioengineering

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