Name: subtype-specific optical action potential recordings in human induced pluripotent stem cell-derived ventricular cardiomyocytes
Uploaded: 2018-09-27T13:30:00
Description: Watch this Scientific Journal Video about Subtype-specific Optical Action Potential Recordings in Human Induced Pluripotent Stem Cell-derived Ventricular Cardiomyocytes at JoVE.com

This work was supported by grants from the German Research Foundation (Si 1747/1-1), the Else Kr&#246;ner-Fresenius-Stiftung, and the Deutsche Stiftung f&#252;r Herzforschung.

1. Preparation of iPSC-derived Cardiomyocytes for Imaging
NOTE: Methods for iPSC culture and cardiac differentiation have been published before12,13,14 and are not discussed here in detail. The purification of iPSC-CMs by manual microdissection, magnetic cell separation, or lactate selection is recommended, depending on the differentiation protocol used. For the following protocol, microdissected explan...

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D., 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=Human+iPSC-derived+cardiomyocytes+cultured+in+3D+engineered+heart+tissue+show+physiological+upstroke+velocity+and+sodium+current+density.">Human iPSC-derived cardiomyocytes cultured in 3D engineered heart tissue show physiological upstroke velocity and sodium current density.</a> Scientific Reports. 7, 5464 (2017).</li><li>Dorn, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+nkx2-5+transcriptional+repression+of+isl1+controls+cardiomyocyte+subtype+identity.">Direct nkx2-5 transcriptional repression of isl1 controls cardiomyocyte subtype identity.</a> Stem Cells. 33 (4), 1113-1129 (2015).</li><li>Kaestner, L., 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=Genetically+Encoded+Voltage+Indicators+in+Circulation+Research.">Genetically Encoded Voltage Indicators in Circulation Research.</a> International Journal of Molecular Sciences. 16 (9), 21626-21642 (2015).</li></ol>

The method described here enables an optical recording of APs from a specific subtype (i.e., ventricular-like cells) of CMs generated from human iPSCs. Human iPSC-CMs are an emerging tool to address a huge variety of biological and medical problems, and the differentiation to different CM subtypes is an important source of experimental variability. By using specific promoter elements, the expression of a GEVI is specifically achieved in CMs representing the subtype of interest, which are then optically imaged.</...

Cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPSCs) are an emerging tool to dissect molecular mechanisms of heart disease, to investigate novel therapies, and to screen for adverse cardiac drug effects1,2,3. Right from the start, arrhythmogenic diseases such as channelopathies have been an important focus of this research area4. Consequently, methods to investigate electrical phenotypes of CMs, such as arrhythmias or changes in action potential (AP) morphologies, are at the heart of this technology.
An important consideration in the application of iPSC-CMs is that current cardiac differentiation protocols do not result in a homogenous population of cells. Instead, they are rather a mixture of cells resembling sinus node, atrial, and ventricular CMs at different levels of maturation5,6,7,8. This heterogeneity can be a relevant source of experimental variability, especially if parameters such as AP duration (APD) are investigated, which intrinsically differ between CM subtypes (e.g., the APD is shorter in atrial than in ventricular CMs). The conventional approach to address this problem is to investigate single iPSC-CMs using the patch clamp method and to classify each cell as nodal-, atrial-, or ventricular-like, based on its AP morphology9. Any subsequent analysis can be then restricted to the cells representing the CM subtype of interest. The major drawback of this strategy is its limited throughput and lack of scalability. Moreover, the invasive nature of patch clamp electrophysiology does not allow the imaging of the same cells sequentially over extended time periods.
Here, we provide experimental details on a method10 developed to optically image APs in specific subtypes of iPSC-CMs. This overcomes the problem of subtype heterogeneity and dramatically increases the throughput as compared to conventional methods, allowing the rapid phenotyping of iPSC-CMs carrying genetic variants or being exposed to pharmacological agents.
Overview of the subtype-specific optical imaging approach
A genetically-encoded voltage indicator (GEVI), whose fluorescence properties change upon depolarization and repolarization of the cell membrane, is used to optically image changes of the membrane potential of CMs. The GEVI applied here is the voltage-sensing fluorescent protein VSFP-CR11, which consists of a voltage-sensing transmembrane domain fused to a pair of a green (Clover) and a red (mRuby2) fluorescent protein (Figure 1A). Due to the close proximity of the two fluorophores, the excitation of the green fluorescent protein results in a fraction of the excitation energy being transferred to the red fluorescent protein via F&#246;rster resonance energy transfer (FRET). Therefore, the excitation of the green fluorescent protein results in an emission from both the green and the red fluorescent proteins (Figure 1A, upper panel). When the cell depolarizes, a structural rearrangement of the voltage sensor occurs that translates into a reorientation of the two fluorescent proteins, increasing the FRET efficiency. Thus, even more of the excitation energy is transferred from the green to the red fluorescent protein (Figure 1A, lower panel). As a result, in a depolarized cell, the green fluorescence emission is dimmer, and the red fluorescence emission is brighter than in a cell at resting membrane potential (Figure 1B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58134/58134fig1.jpg" /> 
Figure 1: Optical imaging of membrane potential with VSFP-CR. (A) A schematic depicting the action of the voltage-sensitive fluorescent protein VSFP-CR is shown. Upon the depolarization of the cell membrane, a structural rearrangement in the voltage-sensing transmembrane domain translates into a reorientation of the green (GFP) and red (RFP) fluorescent protein, increasing the efficiency of the intramolecular F&#246;rster resonance energy transfer (FRET). (B) The emission spectra of a VSFP upon excitation of the GFP in cells at the resting membrane potential (upper panel) and in depolarized cells (lower panel) are depicted. The spectral change upon depolarization is exaggerated for clarity. <a href="https://www.jove.com/files/ftp_upload/58134/58134fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The changes in the FRET efficiency mirroring the fluctuations of the membrane potential are imaged using a fluorescence microscope equipped with an image splitter, which separates red and green fluorescence emissions and projects them onto two adjacent areas of the chip of an sCMOS camera (Figure 2). With this set-up, the fluorescence emission at two different wavelength bands can be recorded simultaneously, which allows the calculation of a ratio of red-to-green fluorescence to reflect the membrane potential in every image of a time-lapse series.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58134/58134fig2.jpg" /> 
Figure 2: Configuration of the imaging system. The principal components of the imaging system used to image the spectral changes of the voltage-sensitive fluorescent protein mirroring the membrane potential changes at a high temporal resolution are depicted. <a href="https://www.jove.com/files/ftp_upload/58134/58134fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The expression of VSFP-CR in CMs is achieved by lentiviral transduction. To direct expression to the CM subtype of interest, the lentivirus contains a promoter element (the MLC2v enhancer) that specifically drives transcription in ventricular-like iPSC-CMs10. When the iPSC-CMs that represent a mixture of atrial-like, nodal-like, and ventricular-like cells are transduced with this lentivirus, VSFP-CR is expressed only in the ventricular-like cells. Since the optical action potential imaging depends on this fluorescent sensor, the recorded action potentials exclusively represent the CM subtype of interest (Figure 3).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58134/58134fig3.jpg" /> 
Figure 3: Promoter-driven VSFP expression for subtype-specific membrane potential imaging. (a)&#160;This schematic shows how cardiomyocyte subtype-specific optical action potential recordings are achieved. (b) iPSC-CMs infected with a VSFP under the control of the ventricular-specific MLC2v-enhancer are shown. The expression of the voltage sensor is observed only in ventricular-like CMs in the GFP channel (left panel). The phase contrast (middle panel) and overlay image (right panel) are also provided. The white dotted lines mark cell boundaries. <a href="https://www.jove.com/files/ftp_upload/58134/58134fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table>
	<tbody>
		<tr>
			<td>&#223;-Mercaptoethanol</td>
			<td>Invitrogen</td>
			<td>21985023</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM-F12 Medium</td>
			<td>Invitrogen</td>
			<td>21331046</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS(Fetal Bovine Serum)</td>
			<td>Invitrogen</td>
			<td>16141079</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids</td>
			<td>Invitrogen</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMax-I Supplement</td>
			<td>Invitrogen</td>
			<td>35050061</td>
			<td>alternative L-Glutamine</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin bovine plasma</td>
			<td>Sigma-Aldrich</td>
			<td>F1141&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase type II</td>
			<td>Worthington Biochem</td>
			<td>LS004174</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexadimethrine Bromide (Polybrene)</td>
			<td>Sigma-Aldrich</td>
			<td>H9268</td>
			<td>enhancing lentiviral infection</td>
		</tr>
		<tr>
			<td>3.5 cm glass-bottom microdishes</td>
			<td>MatTek corporation, Ashland, MA, USA</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope stand</td>
			<td>Leica Microsystems, Wetzlar, Germany</td>
			<td>DMI6000B</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope objective</td>
			<td>Leica Microsystems, Wetzlar, Germany</td>
			<td colspan="2">HCX PL APO 63x/1.4-0.6 Oil</td>
		</tr>
		<tr>
			<td>sCMOS camera</td>
			<td>Andor Technology, Belfast, UK</td>
			<td>Zyla V</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope filter cube: excitation filter</td>
			<td>Chroma Technology Corp, Bellows Falls, VT, USA</td>
			<td>ET480/40X</td>
			<td>bandpass 480/40</td>
		</tr>
		<tr>
			<td>Microscope filter cube: dichroic mirror</td>
			<td>Chroma Technology Corp, Bellows Falls, VT, USA</td>
			<td>T505lpxr</td>
			<td>longpass 505 nm</td>
		</tr>
		<tr>
			<td>Image splitter</td>
			<td>&#160;Cairn Research, Faversham, UK</td>
			<td>OptoSplit II</td>
			<td></td>
		</tr>
		<tr>
			<td>Image splitter filter cube: dichroic mirror</td>
			<td>AHF Analysentechnik GmbH, T&#252;bigen, Germany</td>
			<td>568LPXR</td>
			<td>longpass 568 nm</td>
		</tr>
		<tr>
			<td>Image splitter filter cube: emission filter 1 (GFP emission)</td>
			<td>AHF Analysentechnik GmbH, T&#252;bigen, Germany</td>
			<td>520/28 BrightLine HC</td>
			<td>bandpass 520/28 nm</td>
		</tr>
		<tr>
			<td>Image splitter filter cube: emission filter 2 (RFP emission)</td>
			<td>AHF Analysentechnik GmbH, T&#252;bigen, Germany</td>
			<td>630/75 ET Bandpass</td>
			<td>bandpass 630/75 nm</td>
		</tr>
		<tr>
			<td>Pacing inset</td>
			<td>Warner Instruments, Hamden, CT, USA</td>
			<td>RC-37FS</td>
			<td></td>
		</tr>
	</tbody>
</table>

subtype-specific optical action potential recordings in human induced pluripotent stem cell-derived ventricular cardiomyocytes

Cardiomyocytes generated from human induced pluripotent stem cells (iPSC-CMs) are an emerging tool in cardiovascular research. Rather than being a homogenous population of cells, the iPSC-CMs generated by current differentiation protocols represent a mixture of cells with ventricular-, atrial-, and nodal-like phenotypes, which complicates phenotypic analyses. Here, a method to optically record action potentials specifically from ventricular-like iPSC-CMs is presented. This is achieved by lentiviral transduction with a construct in which a genetically-encoded voltage indicator is under the control of a ventricular-specific promoter element. When iPSC-CMs are transduced with this construct, the voltage sensor is expressed exclusively in ventricular-like cells, enabling subtype-specific optical membrane potential recordings using time-lapse fluorescence microscopy.

In Figure 4a, a representative single iPSC-CM is depicted with the white dotted lines marking the ROI drawn during the imaging analysis in the RFP (left side) and the GFP (right side) channel. The signal from the RFP channel shows a periodic increase in fluorescent intensity during each action potential (Figure 4b, upper panel). As described in the Introduction, this is due to an increasing FRET caused by changes in the membrane ...

Watch this Scientific Journal Video about Subtype-specific Optical Action Potential Recordings in Human Induced Pluripotent Stem Cell-derived Ventricular Cardiomyocytes at JoVE.com

Subtype-specific Optical Action Potential Recordings in Human Induced Pluripotent Stem Cell-derived Ventricular Cardiomyocytes

Here we present a method to optically image action potentials, specifically in ventricular-like induced pluripotent stem cell-derived cardiomyocytes. The method is based on the promoter-driven expression of a voltage-sensitive fluorescent protein.

Cardiomyocytes generated from human induced pluripotent stem cells (iPSC-CMs) are an emerging tool in cardiovascular research. Rather than being a ...

Cardiomyocytes generated from Induced Pluripotent Stem or IPS cells are increasingly used to answer a variety of biological questions in the field of cardiac electrophysiology. The main advantages of this technology are that it allows a dramatic increase in throughput as well as subtype specific action potential recordings in ventricular like myocytes, using a ventricular specific promoter element. The introduction of genetically encoded voltage sensors through lentiviral transduction allows the optical imaging of human-induced pluripotent stem cells-derived cardiomyocytes action potentials.Begin by mixing lentivirus-containing cell culture supernatant with cardiomyocyte maintenance medium, at a one to one ratio. Add hexadimetherine bromide at a final concentration of 8 micrograms per milliliter, to enhance the infection efficiency. And replace the cardiomyocyte maintenance medium in the IPS cell-derived cardiomyocytes culture dish, with the infection medium.Culture the infected cardiomyocytes for 12 hours at 37 degrees Celsius, and replace the infection medium with fresh cardiomyocyte maintenance medium. Before imaging, exchange the cell culture medium with Tyrode's solution, supplemented with calcium. Then place the cardiomyocytes in an imaging chamber on the stage of an inverted epifluorescence microscope equipped with an image splitter.If electrical pacing is required, install pacing electrodes in the imaging chamber and connect the electrodes to a stimulus generator. Bring the cells into focus, and locate a cell expressing the genetically encoded voltage indicator in the center of the viewing field. Set the optical path so that the emitted light is routed to the image splitter and switch the filter cube in the microscope to the one to be combined with the filters in the image splitter.In the software controlling the camera, set the acquisition setting so that high-speed imaging is possible, and set up the image splitter according to the manufacturer's instructions. The two images representing the two different emission wavelength bands should be adjacent to each other, each occupying one half of the image. Check and adjust the focus of the image produced by the camera as necessary.And adjust the brightness of the illumination light so that pixel saturation is avoided. Set the acquisition of the time series, and dim the light in the room to avoid stray light influencing the measurement. Then open the excitation light shutter and begin the acquisition of the image series, starting the electrical pacing at the appropriate time point, as experimentally appropriate.When the acquisition is finished, close the excitation light shutter as necessary and save the recording to the hard drive. Then, leaving the recording setting unchanged, record the background over a region of the coverslip without cells. For analysis of the optical membrane potential recordings, open the tiff stack containing the cells in ImageJ, and use the free-hand selection tool to draw a region of interest over a fluorescent cardiomyocyte, in the red channel, taking care that the area is large enough that the cell will not move out of the region while contracting.Next, select Analyze, Tools, Region Of Interest Manager and select Add T, to add the region as Region Of Interest 1. Drag the region of interest over the same cell in the green channel, and add this region as Region Of Interest 2. Under the Analyze and Set Measurements menus, unselect all of the options except for the Mean gray value.In the Region Of Interest Manager, click More and Multi Measure to select the Measure all slices and One row per slice options and click OK.A window will open containing a spreadsheet with the three rows. Use File and Save As to save the spreadsheet into a comma separated value file. Close the window with the image stack and the spreadsheet window without closing the Region of Interest Manager window, and open the tiff stack containing the background measurement.In the Region Of Interest Manager, click the More and Multi Measure to select the Measure all slices and One row per slice options and click OK.Then save the background data in a separate csv file. Here, a representative single IPS cell-derived cardiomyocyte is depicted with the white dotted lines marking the regions of interest, drawn during the imaging analysis in the RFP and GFP channels is shown. The signal from the RFP channel demonstrates a periodic increase in fluorescent intensity during each action potential due to an increasing forced or resonance energy transfer caused by changes in the membrane potential.Concordantly, the GFP signal exhibits a periodic decrease in fluorescent intensity. The RFP/GFP ratio is the biological signal used in downstream analyses, such as action potential duration 50 and 90 measurements. For example, using this method, 30-day old ventricular like IPS cell-derived cardiomyocytes placed at 0.5 Hertz, showed a mean action potential duration 50 of 439 milliseconds, and a mean action potential duration 90 of 520 milliseconds.Electrical stimulation of the IPS cell-derived cardiomyocytes at increasing stimulation rates every five beats, demonstrated a progressive shortening of the action potentials as the beating rate increased. Moreover, treatment of the spontaneously beating IPS cell-derived cardiomyocytes with isoproternol, a nonselective beta adreno receptor agonist, led to a prompt increase in the beating rate, that could be further increased with increasing doses of the agonist. While this method provides superior throughput compared to classical electrophysiological measurements, one must be aware of its limitations, including a temporal resolution that is limited by the intrinsic kinetic properties of the fluorescent voltage sensor.

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Biology

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...

Automated Production of Human Induced Pluripotent Stem Cell-Derived Cortical and Dopaminergic Neurons with Integrated Live-Cell Monitoring

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are prone to spontaneous differentiation into unwanted cell types. The methods are labor-intensive and are not easily amenable to large-scale experiments. To overcome these limitations, we developed an automated cell culture system coupled to a high-throughput imaging system and implemented protocols for maintaining multiple hiPSC lines in parallel and neuronal differentiation. We describe the automation of a short-term differentiation protocol using Neurogenin-2 (NGN2) over-expression to produce hiPSC-derived cortical neurons within 6&#8210;8 days, and the implementation of a long-term differentiation protocol to generate hiPSC-derived midbrain dopaminergic (mDA) neurons within 65 days. Also, we applied the NGN2 approach to a small molecule-derived neural precursor cells (smNPC)&#160;transduced with GFP lentivirus and established a live-cell automated neurite outgrowth assay. We present an automated system with protocols suitable for routine hiPSC culture and differentiation into cortical and dopaminergic neurons. Our platform is suitable for long term hands-free culture and high-content/high-throughput hiPSC-based compound, RNAi and CRISPR/Cas9 screenings to identify novel disease mechanisms and drug targets.

We show the automation of human induced pluripotent stem cell (hiPSC) cultures and neuronal differentiations compatible with automated imaging and analysis.

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are ...

Real-Time Measurements of Calcium and Contractility Parameters in hiPSC-CMs

Real-Time Measurements of Calcium and Contractility Parameters in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a powerful tool for studying mutation-mediated changes in cardiomyocyte function and defining the effects of stressors and drug interventions. In this study, it is demonstrated that this optics-based system is a powerful tool to assess the functional parameters of hiPSC-CMs in 2D. By using this platform, it is possible to perform paired measurements in a well-preserved temperature environment on different plate layouts. Moreover, this system provides researchers with instant data analysis.
This paper describes a method for measuring the contractility of unmodified hiPSC-CMs. Contraction kinetics are measured at 37 &#176;C based on pixel correlation changes relative to a reference frame taken at relaxation at a 250 Hz sampling frequency. Additionally, simultaneous measurements of intracellular calcium transients can be acquired by loading the cell with a calcium-sensitive fluorophore, such as Fura-2. Using a hyperswitch, ratiometric calcium measurements can be performed on a 50 &#181;m diameter illumination spot, corresponding to the area of the contractility measurements.

Author Spotlight: Real-Time Measurements of Calcium and Contractility Parameters in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes  

Here, an established method is described to perform contractility and calcium measurements in human induced pluripotent stem cell-derived cardiomyocytes using an optics-based platform. This platform enables researchers to study the effect of mutations and the response to various stimuli in a rapid and reproducible manner.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a powerful tool for studying mutation-mediated changes in cardiomyocyte ...