The authors want to thank the members of the ICREC Research Program (IGTP, Badalona) and the Electronic and Biomedical Instrumentation Group (UPC, Barcelona), especially Prof. J. Rosell-Ferrer. In addition, the authors acknowledge STEM CELLS Translational Medicine journal and AlphaMed Press for permitting the adaptation of previously published figures (Lluci&#224;-Valldeperas, et al.30). The development of this prototype and the design of the protocol were supported by Ministerio de Educaci&#243;n y Ciencia (SAF 2008-05144), Ministerio de Econom&#237;a y Competitividad (SAF 2014-59892), the European Commission 7th Framework Programme (RECATABI, NMP3-SL-2009-229239), Fundaci&#243; La Marat&#243; de TV3 (080330, 201516, 201502), and Fundaci&#243;n para la Innovaci&#243;n y la Prospectiva en Salud en Espa&#241;a (FIPSE; 06-00001396-15).

This study uses human cardiac ATDPCs from patient samples. Their use has been approved by the local ethics committee, and all patients gave informed consent. The study protocol conforms to the principles outlined in the Declaration of Helsinki.
1. Preparations
<ol>
	<li>Autoclave two tweezers, 12 platinum PTFE electrodes for electrical stimulation, and some paper towels, at 121 &#176;C for 20 min.</li>
	<li>Sterilize 12 PDMS custom-made constructs (Figure 1A).
	<ol>
		<li>Wash each construct with 5 mL of sterile, distilled water with magnetic agitation at room temperature for 15 min.</li>
		<li>Wash 1x with 5 mL of 70% ethanol with magnetic agitation at room temperature for 5 min.</li>
		<li>Wash 5x with 5 mL of sterile, distilled water with magnetic agitation at room temperature for 10 min per wash, to remove alcoholic residues.</li>
		<li>Dry the constructs on sterile paper towels inside the flow cabinet overnight.</li>
		<li>Store them in sterile 50 mL centrifuge tubes until use.</li>
	</ol>
	</li>
</ol>
2. Cell Seeding (Day -1)
<ol>
	<li>Before cell seeding, transfer the cleaned PDMS constructs to sterile plates and expose them to ultraviolet light for 5 min to ensure complete sterilization.</li>
	<li>Transfer each construct to a 35 mm cell culture plate, or 6-well plate,&#160;for immediate cell seeding.</li>
	<li>Trypsinize a confluent T75 flask of cardiac ATDPCs.
	<ol>
		<li>Wash the T75 flask with 5 mL of 1x phosphate-buffered saline (PBS).</li>
		<li>Add 1 mL of 0.05% trypsin-EDTA and incubate at 37 &#176;C for 5 min to detach the cells.</li>
		<li>Add 5 mL of complete medium to inactivate the trypsin-EDTA.</li>
		<li>Collect all cells in a 15 mL tube and wash the flask 2x with 5 mL of PBS to collect any remaining cells.</li>
		<li>Centrifuge at 230 x g for 5 min at 22 &#176;C, remove the supernatant, and resuspend the cells in 2 mL of complete medium to count them with the hemocytometer chamber.</li>
	</ol>
	</li>
	<li>Seed 200 &#181;L of cardiac ATDPCs (2.5 x 105 cells/mL) into the cell pool of the 12 PDMS constructs (Figure 1A,B) to have ~80% of the seeding surface covered by cells the day after, and incubate at 37 &#176;C and 5% CO2. 
	NOTE: After 2&#8211;4 h, the cells should be attached. Cell inoculum is prepared according to the cell size and growth. For smaller cells, the seeding density should be increased.</li>
	<li>Gently add 2 mL of prewarmed complete medium (&#945;-MEM supplemented with 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin-streptomycin) per plate.</li>
	<li>Incubate the constructs at culture conditions (usually 37 &#176;C and 5% CO2) overnight.</li>
</ol>
3. Electromechanical Stimulation Setup (Day 0)
<ol>
	<li>Before starting the procedure, take six constructs for electromechanical stimulation and six as nonstimulated controls. With fewer than six plates per stimulation, use empty constructs with the same medium volume to ensure proper electric field stimulation.</li>
	<li>Clean the stimulation unit with 70% ethanol and place it in the flow cabinet.</li>
	<li>Bring the sterile electrodes and tweezers inside the flow cabinet.</li>
	<li>Remove 90% of the media from the culture plate to easily manipulate the electrodes and constructs. First, place the PDMS constructs in the right position to ensure magnetic attraction (PDMS displacement within the culture plate toward the magnet) between both fixed and mobile magnets. Next, connect the platinum wire to the electrode connectors and the PTFE part in its designated space in the PDMS construct.</li>
	<li>Add 2.5 mL of fresh prewarmed complete medium to each construct. 
	NOTE: Maintain the sterility throughout the procedure and operate on one construct at a time. Maintain the rest of the constructs in the incubator at 37 &#176;C and 5% CO2 until use.</li>
	<li>Once all PDMS constructs are placed and electrically connected to the platform, bring the platform back into the incubator at 37 &#176;C and 5% CO2.</li>
	<li>Connect the electrical and mechanical source.</li>
	<li>Configure the stimulation program. Specify electrical and mechanical stimulation regimes through the user interfaces of the electrical stimulator and the application, which controls the mechanical stimulation. Set the synchronism as follows.
	<ol>
		<li>Switch on the electrical stimulator. Wait for the main menu to appear in the display.
		<ol>
			<li>Then, select Option 2: Edit sequence + Enter.</li>
			<li>Edit the sequence Menu as follows.
			<ol>
				<li>Use the Mode tab to select either voltage or current. Select current by clicking + and press Enter.</li>
				<li>For the Amplitude tab, select 1 (mA) with +/- and press Enter.</li>
				<li>For the Period (T), select 1000 (ms) with +/- and press Enter.</li>
				<li>Set the Pulse duration (Tw) to 2 (ms) with +/- and press Enter.</li>
				<li>For the Trigger mode tab, select External by software and press Enter.</li>
				<li>Back in the main menu, select Option 4: Generate sequence and press Enter. 
				NOTE: The electrical stimulator rests in the standby until it receives a trigger command from the mechanical stimulator application through the serial port.</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>Perform the following steps in the mechanical stimulation section of the control application panel (Figure 2C).
		<ol>
			<li>Write 1,000 (ms) in the Pulse Period text control.</li>
			<li>Write 500 (ms) in the ON time (Tw) text control to set the mechanical pulse duration.</li>
			<li>Write 2,000 (AU) in the Excursion text control to deliver a 10% construct elongation. This is the number of steps in the linear control motor. 
			NOTE: The stimulation protocol applied here consists of alternating-current 2 ms monophasic square-wave pulses of 50 mV/cm at 1 Hz and 10% stretching for 7 days. The rise and fall times of the mechanical pulse are set at 100 ms, to roughly imitate the shape of the hearth pressure pulse. Also, the repetition mode is set to Continuous and there is a counter displaying the number of pulses.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Change the media 2x a week (Monday and Thursday afternoon). First, remove the old media; next, add the warm media on the sides of the PDMS support, never directly on the cell pool. 
	NOTE: If the cells have a high growth rate, the media should be changed 3x a week (e.g., Monday, Wednesday, and Friday). It is necessary to disconnect and reconnect all cables, but there is no need to remove the culture plates and electrodes from their place.</li>
	<li>Collect the samples after the experimentation is performed.</li>
</ol>
4. Sample Collection at the End of the Experimentation (Day 7)
<ol>
	<li>For RNA analyses
	<ol>
		<li>Wash the construct 2x with 3 mL of 1x PBS for 5 min at room temperature.</li>
		<li>Add 3 mL of 0.05% trypsin-EDTA to each plate (enough to cover the whole construct) and wait for 5 min at 37 &#176;C.</li>
		<li>After the cells are detached, add 2 mL of complete medium to inactivate the trypsin-EDTA.</li>
		<li>Collect all cells in a 15 mL tube and wash the construct 2x with 3 mL of PBS to collect any remaining cells.</li>
		<li>Centrifuge at 230 x g for 5 min at 22 &#176;C.</li>
		<li>Remove the supernatant and resuspend the pellet in 1 mL of PBS.</li>
		<li>Transfer the cell solution to a 1.5 mL tube and centrifuge at 230 x g for 5 min.</li>
		<li>Remove the supernatant and store the pellet at -80 &#176;C in 700 &#181;L of lysis reagent for further RNA isolation.</li>
		<li>Isolate RNA using a commercial kit, following the manufacturer&#39;s instructions.</li>
		<li>Reverse-transcribe the isolated RNA using the kit and random hexamers, according to the manufacturer&#39;s protocol.</li>
		<li>For small RNA concentrations, preamplify and, then, dilute 1:5 with RNase-free water before subsequent real-time reverse transcription polymerase chain reaction (RT-PCR) is performed. Continue with the standard protocol for real-time RT-PCR and check main cardiac markers. 
		NOTE: Typical cardiac markers comprise early and late markers from different categories, such as cardiac transcription factors (myocyte-specific enhancer factor 2A [MEF2A], GATA-binding protein 4 [GATA-4]) and structural (cardiac troponin I [cTnI], cardiac troponin T [cTnT], &#945;-actinin) and calcium regulation (Connexin43 [Cx43], sarco-/endoplasmic reticulum Ca2+-ATPase [SERCA2])30. Protein isolation can also be performed if needed. Simultaneous RNA and protein isolation can be performed with the same sample, using commercially available reagents and kits (Table of Materials) if the sample quantity is less.</li>
	</ol>
	</li>
	<li>For immunostainings 
	NOTE: This is performed directly on the cells attached to the cell pool of the PDMS construct. Therefore, we recommend placing, every time, 1 cm x 1 cm of paraffin film on the top of the cell pool, apart from the plate lid, to minimize the evaporation of the incubation solutions.
	<ol>
		<li>Wash the construct 2x with 3 mL of PBS for 5 min at room temperature.</li>
		<li>Fix the cells attached to the construct with 2 mL of 10% formalin for 15 min at room temperature.</li>
		<li>Wash the cells 3x with 3 mL of PBS for 5 min at room temperature. For long-term storage, leave the samples in PBS with 0.1% sodium azide at 4 &#176;C.</li>
		<li>Permeabilize the cells with 3 mL of PBS + 0.5% detergent (3x, each time 5&#8211;10 min, at room temperature).</li>
		<li>Incubate the cells with 100 &#181;L of PBS + 10% horse serum + 0.2% detergent + 1% bovine serum albumin at room temperature for 1 h, to block nonspecific antibody binding.</li>
		<li>Incubate the cells with 100 &#181;L of PBS + 10% horse serum + 0.2% detergent + 1% bovine serum albumin + primary antibody at room temperature for 1 h. For example, primary antibodies against Cx43 (1:100), sarcomeric &#945;-actinin (1:100), GATA-4 (1:50), MEF2 (1:25), and SERCA2 (1:50).</li>
		<li>Wash 3x with 3 mL of PBS at room temperature for 5 min.</li>
		<li>Incubate the cells with 100 &#181;L of PBS + secondary antibody at room temperature in the dark for 1 h. 
		NOTE: Secondary antibodies conjugated with different fluorophores and a counterstaining agent were used.</li>
		<li>Wash them 3x with 3 mL of PBS at room temperature in the dark for 5 min.</li>
		<li>Incubate the cells with 100 &#181;L of nuclear staining (0.1 &#181;g/mL) in PBS at room temperature in the dark for 15 min.</li>
		<li>Wash them 3x with 3 mL of PBS at room temperature in the dark for 5 min.</li>
		<li>Store the samples in 3 mL of PBS with 0.1% sodium azide at 4 &#176;C until the acquisition. 
		NOTE: Microscope acquisition is possible on inverted fluorescent and confocal microscopes with long-working distance objectives because the construct thickness is about 0.5 mm.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose, except that the stimulation device and protocol were previously patented (WO-2013185818-A1, WO-2017125159-A1).

Electromechanical stimulation appears to be a safe alternative for preparing cells for a hostile cardiac environment and enhancing their cardiac commitment. Here, a protocol described for cardiac progenitor cells increased the expression of main cardiac markers and was reported to be beneficial for their next implantation on infarcted murine myocardium30. In general, electromechanically stimulated cardiac ATDPCs increased the expression of genes related to early, structural, and calcium regulation...

Heart function is based on the coupling of electrical excitation and mechanical contraction. Briefly, cardiomyocyte intercellular junctions permit electrical signal propagation to produce almost synchronous contractions of the heart that pump blood systemically and through the pulmonary system. Cardiac cells, thus, undergo both electrical and mechanical forces that regulate gene expression and cellular function. Accordingly, many groups have attempted to develop culture platforms that mimic the cardiac physiological environment to understand the role of mechanical and electrical stimulation on cardiac development, function, and maturation. In vitro electrical and mechanical stimulations individually have been applied extensively in cardiac tissue engineering to enhance functional properties, increase cell maturation, or improve cell-cell coupling and calcium handling1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21. Nevertheless, synchronous electromechanical conditioning remains unexploited because of the challenge of developing a stimulator and protocol, and because of the mandatory optimization22.
Preliminary work addressed electromechanical stimulation as a combination of electrical stimulation and media perfusion; however, the flow does not involve the strain-based deformation typical of ventricular filling23,24,25. Later, more physiological approaches combined electrical stimuli with physical deformation or stretch to mimic the isovolumetric contraction26,27,28,29,30,31. Feng et al. described the first demonstration of electromechanical stimulation in 2005, reporting enhanced cardiomyocyte size and contractile properties26. Wang et al. pretreated mesenchymal stem cells with 5-azacytidine and applied simultaneous electrical and mechanical conditioning, improving recellularization, cell viability, cardiac differentiation, and tissue remodeling27. Since those publications, more groups have reported on electromechanical stimulation of cell monolayers or engineered tissues (e.g., Black28, Vunjak-Novakovic29,31, and our group30) with the first conditioned cells tested in vivo30. Briefly, Morgan and Black tested several combinations of electrical and mechanical stimuli, reporting that the timing between stimulations was crucial because delayed combined electromechanical stimulation yielded the best results28. Next, Godier-Furn&#233;mont and collaborators optimized an electromechanical stimulation protocol for engineered heart muscle constructs from neonatal rat heart cells and achieved, for the first time, a positive force-frequency relationship29. Afterward, our group reported that electromechanically preconditioned cells increased the expression of main cardiac markers in vitro and broad beneficial effects in vivo, such as improved cardiac function or increased vessel density in the infarct border region30. The most recent publication demonstrated that cardiac tissues from stem-cell-derived cardiomyocytes subjected to electromechanical conditioning reached a maturation level closer to human adult cardiac structure and function31. Additionally, alternative three-dimensional stimulation platforms comprise electroactive scaffolds that provide electrical, mechanical, and topographical cues to the cells attached32. Moreover, mechanical deformation (cell monolayer stretching and compression) can also be induced with stretchable electrodes mimicking normal physiological conditions, as well as extreme conditions33.
Therefore, the rationale is that in vitro electromechanical stimuli based on physiological conditions could enhance the cardiomyogenic potential of a cell. Indeed, this stimulation could benefit further integrations of therapeutic cells into the myocardium in a clinical scenario or increase tissue maturation for drug-screening applications.
In addition, we isolated and characterized a population of human adipose tissue-derived progenitor cells of cardiac origin (cardiac ATDPCs)34. These cells are located in the epicardial fat. These cells display beneficial histopathological and functional effects in the treatment of myocardial infarction and also maintain cardiac and endothelial differentiation potential.30,35. We hypothesized that these benefits would increase after biophysical stimulation.
Consequently, we developed a device and a stimulation regime for the cell population of interest and investigated the effects. This electromechanical protocol is a new strategy to induce active cell stretching in a sterile manner and noninvasively compared to previous publications36, in combination with electric field stimulation. The technique reported here explains in detail the device and method used for the electrical, mechanical, and electromechanical stimulation of cells.
This device can provide both electrical and mechanical stimulation, independently or simultaneously. The stimulation is performed with a noninvasive and aseptic novel approach that includes presterilized cell support, electrodes placed inside a standard culture plate, and a platform that induces the mechanical and electrical forces (Figure 1).
The platform can hold up to six culture plates and consists of a sandwich structure of laser-cut poly(methyl methacrylate) and printed circuit-board pieces. The platform prototype relies on a combination of a monophasic programmable computer-controlled electrical stimulator, a printed circuit board for the robust connection of the electrodes, and six 10 mm x 10 mm x 5 mm nickel-plated neodymium-fixed magnets placed near one side of the culture plates. There is also an aluminum bar with six driving magnets (same model) placed in front of the other side of the culture plates and moved with a linear servomotor. The motor is driven by a motor controller, operated through an RS-232 port by commercial software (see the Table of Materials). Through the user interface and programmable stimulator, it is possible to program the electrical intensity, the pulse duration and frequency, the frequency of mechanical stimulation, its duty cycle, the number of pulses, the pulse amplitude (magnet excursion), and the slope.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58934/58934fig1.jpg" /> 
Figure 1: Electromechanical stimulator. (A) PDMS construct used for the cell conditioning. (B) Drawing of the PDMS construct, including electrodes and magnets. (C) Detail of the printed circuit board (platform) used to perform the electromechanical conditioning. This panel has been modified from Lluci&#224;-Valldeperas et al.30. (D) Picture of the electromechanical stimulation platform and user interface (computer). <a href="https://www.jove.com/files/ftp_upload/58934/58934fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Both the stimulator and the method for electromechanical conditioning are fully described in two international patents, WO-2013185818-A137 and WO-2017125159-A138.
The biocompatible silicone constructs designed to provide structural support to cells, electrodes, and magnets have been described previously10,21. Briefly, they consist of polydimethylsiloxane (PDMS), molded and cured at room temperature, with a Young&#39;s modulus of 1.3 MPa, close to physiological levels. The construct contains a cell culture pool in a flexible area (10 mm x 10 mm x 2 mm), two inner transverse slots to hold the electrodes, and two embedded 6 mm x 2 mm x 4 mm nickel-plated neodymium magnets. The electrodes are built with 0.2 mm platinum wire twisted around a 2 mm x 3 mm x 12 mm polytetrafluoroethylene (PTFE) core bar (21 cm per electrode, approximately 23 turns) and placed at opposite sides of the flexible area to create an electric field for inducing electrical stimulation. Mechanical stretching is achieved through magnetic attraction between magnets embedded in the support and external magnets placed next to the culture plate and on the moving aluminum arm. In this way, the cell support can be extended without breaking the sterile barrier. This approach is suitable for a cell monolayer but could be adapted to three-dimensional constructs, as well.
In addition, a regular pattern could be imprinted where the cells are seeded, using a ruled diffraction grating (1,250 grooves/mm). The direct visualization of cells cultured on the PDMS construct under brightfield and fluorescent microscopes is possible because of its transparency and 0.5 mm thickness. In the current case, the PDMS culture pool has a vertical surface pattern, perpendicular to the stretching force, to align the cells perpendicularly to the electric field, which minimizes the electric field gradient across the cell.
Figure 1 shows a detailed description of the construct and device used for the stimulation. The PDMS construct and characteristics are optimized for cell stretching (Figure 1A,B). The stimulator is developed and validated for the effective application of the desired electrical and mechanical stimulation to cells attached to the PDMS construct. This process includes ensuring good connectivity and user operability through the software interface (Figure 1C,D).
The procedure for cell stimulation using this custom-made device is described in the protocol section.

<table><tbody><tr><td>&lt;strong&gt;Stimulator&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>nickel plated neodymium magnets</td><td>Supermagnete</td><td>Q-10-10-05-N</td><td></td></tr><tr><td>nickel-plated neodymium magnets</td><td>Supermagnete</td><td>Q-06-04-02-HN</td><td></td></tr><tr><td>polydimethylsiloxane (PDMS) SYLGAR 184 Silicone Elastomer Kit</td><td>Dow Corning Corp</td><td>184</td><td></td></tr><tr><td>ruled diffraction grating (1250 grooves/mm)</td><td>Newport</td><td>05RG150-1250-2</td><td></td></tr><tr><td>Motor controller</td><td>Faulhaber</td><td>MCLM-3006-S</td><td></td></tr><tr><td>Labview</td><td>National Instruments</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Cell culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>phosphate-buffered saline (PBS)</td><td>Gibco</td><td>70013-065</td><td></td></tr><tr><td>0.05% trypsin-EDTA</td><td>Gibco</td><td>25300-120</td><td></td></tr><tr><td>35 mm cell culture dish</td><td>BD Falcon</td><td>45353001</td><td></td></tr><tr><td>fetal bovine serum (FBS)</td><td>Gibco</td><td>10270-106</td><td></td></tr><tr><td>L-Glutamine 200 mM, 100x</td><td>Gibco</td><td>25030-024</td><td></td></tr><tr><td>Penicilina/Streptomicine, 10.000 U/mL</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Minimum essential medium eagle (alfa-MEM)</td><td>Sigma</td><td>M4526-24x500ML</td><td></td></tr><tr><td>&lt;strong&gt;Protein &amp;amp; RNA analyses&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>protease inhibitor cocktail</td><td>Sigma</td><td>P8340</td><td></td></tr><tr><td>QIAzol Lysis Reagent</td><td>Qiagen</td><td>79306</td><td></td></tr><tr><td>AllPrep RNA/Protein Kit</td><td>Qiagen</td><td>50980404</td><td></td></tr><tr><td>Rneasy mini kit</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>iTaq Universal Probes One-Step Kit</td><td>Bio-Rad Laboratories</td><td>172-5140</td><td></td></tr><tr><td>Random hexamers</td><td>Qiagen</td><td>79236</td><td></td></tr><tr><td>TaqMan PreAmp MasterMix 2x</td><td>Applied Biosystems</td><td>4391128</td><td></td></tr><tr><td>TaqMan Universal PCR MasterMix</td><td>Applied Biosystems</td><td>4324018</td><td></td></tr><tr><td>&lt;strong&gt;Immunostaining&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10% formalin</td><td>Sigma</td><td>HT-501128-4L</td><td></td></tr><tr><td>horse serum</td><td>Sigma</td><td>H1138</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>X100-500ML</td><td></td></tr><tr><td>Bovine Serum Albumina (BSA)</td><td>Sigma</td><td>A7906-100G</td><td></td></tr><tr><td>PARAFILM</td><td>Sigma</td><td>P6543</td><td></td></tr><tr><td>4&amp;rsquo;,6-diamidino-2-phenylindole (DAPI)</td><td>Sigma</td><td>D9542</td><td></td></tr><tr><td>Phalloidin Alexa 568</td><td>Invitrogen</td><td>A12380</td><td></td></tr><tr><td>sodium azide</td><td>Sigma</td><td>S8032-100g</td><td></td></tr><tr><td>Hoechst 33342</td><td>Sigma</td><td>14533</td><td></td></tr><tr><td>Connexin-43 rabbit primary antibody</td><td>Sigma</td><td>C6219 lot#061M4823</td><td></td></tr><tr><td>sarcomeric &amp;alpha;-actinin mouse primary antibody</td><td>Sigma</td><td>A7811 lot#080M4864</td><td></td></tr><tr><td>GATA-4 goat primary antibody</td><td>R&amp;amp;D</td><td>AF2606 VAZ0515101</td><td></td></tr><tr><td>MEF2 rabbit primary antibody</td><td>Santa Cruz</td><td>sc-313 lot#E0611</td><td></td></tr><tr><td>SERCA2 goat primary antibody</td><td>Santa Cruz</td><td>sc-8095 lot#D2709</td><td></td></tr><tr><td>Cy3 secondary antibody</td><td>Jackson ImmunoResearch</td><td>711-165-152</td><td></td></tr><tr><td>Cy3 secondary antibody</td><td>Jackson ImmunoResearch</td><td>715-165-151</td><td></td></tr><tr><td>Cy3 secondary antibody</td><td>Jackson ImmunoResearch</td><td>712-165-150</td><td></td></tr><tr><td>Cy2 secondary antibody</td><td>Jackson ImmunoResearch</td><td>715-225-150</td><td></td></tr><tr><td>Cy2 secondary antibody</td><td>Jackson ImmunoResearch</td><td>711-225-152</td><td></td></tr><tr><td>Cy2 secondary antibody</td><td>Jackson ImmunoResearch</td><td>705-225-147</td><td></td></tr></tbody></table>

simultaneous electrical and mechanical stimulation to enhance cells' cardiomyogenic potential

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated researchers in the stem cell and bioengineering fields to develop in vitro high-fidelity human myocardium for both basic research and clinical applications. However, the immature phenotype of cardiac cells is a limitation on obtaining tissues that functionally mimic the adult myocardium, which is mainly characterized by mechanical and electrical signals. Thus, the purpose of this protocol is to prepare and mature the target cell population through electromechanical stimulation, recapitulating physiological parameters. Cardiac tissue engineering is evolving toward more biological approaches, and strategies based on biophysical stimuli, thus, are gaining momentum. The device developed for this purpose is unique and allows individual or simultaneous electrical and mechanical stimulation, carefully characterized and validated. In addition, although the methodology has been optimized for this stimulator and a specific cell population, it can easily be adapted to other devices and cell lines. The results here offer evidence of the increased cardiac commitment of the cell population after electromechanical stimulation. Electromechanically stimulated cells show an increased expression of main cardiac markers, including early, structural, and calcium-regulating genes. This cell conditioning could be useful for further regenerative cell therapy, disease modeling, and high-throughput drug screening.

Figure 2 represents the general schema followed for the cell stimulation. Briefly, cells were seeded on the PDMS construct and subjected to electromechanical stimulation, with a media change performed twice a week. Nonstimulated cells were used as a control for the electromechanical conditioning. Additionally, we added an extra control to the experiment, and subcutaneous ATDPCs were used as a control for cardiac ATDPCs. Subcutaneous ATDPCs are obtained from s...

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Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

Here we present a protocol for training a cell population using electrical and mechanical stimuli emulating cardiac physiology. This electromechanical stimulation enhances the cardiomyogenic potential of the treated cells and is a promising strategy for further cell therapy, disease modeling, and drug screening.

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated ...

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7.5K Views.  Health Science Research Institute Germans Trias i Pujol. Dit is een methode om elektromechanische stimulatie op cellen toe te passen. Het heeft meerdere toepassingen om de effecten ervan op een celpopulatie te bestuderen, voortraining voor in vivo levering en celrijping. Het voordeel is dat elektrische en mechanische stimuli individueel of gelijktijdig met hetzelfde apparaat kunnen worden toegepast terwijl steriele virale intact blijven.Deze techniek is een indirecte benadering van onze therapie. Celtherapie wordt beschouwd als elektromechan...

Video: Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

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

Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their potential applications in cardiac research, including drug discovery, disease modeling, tissue engineering, and regenerative medicine. However, cells produced by existing protocols display a range of immaturity compared with native adult ventricular cardiomyocytes. Many efforts have been made to mature hPSC-CMs, with only moderate maturation attained thus far. Therefore, an engineered system, called biowire, has been devised by providing both physical and electrical cues to lead hPSC-CMs to a more mature state in vitro. The system uses a microfabricated platform to seed hPSC-CMs in collagen type I gel along a rigid template suture to assemble into aligned cardiac tissue (biowire), which is subjected to electrical field stimulation with a progressively increasing frequency. Compared to nonstimulated controls, stimulated biowired cardiomyocytes exhibit an enhanced degree of structural and electrophysiological maturation. Such changes are dependent upon the stimulation rate. This manuscript describes in detail the design and creation of biowires.

The cardiac biowire platform is an in vitro method used to mature human embryonic and induced pluripotent stem cell-derived cardiomyocytes (hPSC-CM) by combining three-dimensional cell cultivation with electrical stimulation. This manuscript presents the detailed setup of the cardiac biowire platform.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their ...

Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. Y-27632 is a highly potent inhibitor of Rho-associated, coiled-coil-containing protein kinase (RhoA/ROCK) and is used to prevent dissociation-induced cell apoptosis (anoikis). We demonstrate that Y-27632 pretreatment for human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs+RI) prior to implantation results in a cell engraftment rate improvement in a mouse model of acute myocardial infarction (MI). Here, we describe a complete procedure of hiPSC-CMs differentiation, purification, and cell pretreatment with Y-27632, as well as the resulting cell contraction, calcium transient measurements, and transplantation into mouse MI models. The proposed method provides a simple, safe, effective, and low-cost method which significantly increases the cell engraftment rate. This method cannot only be used in conjunction with other methods to further enhance the cell transplantation efficiency but also provides a favorable basis for the study of the mechanisms of other cardiac diseases.

In this protocol, we demonstrate and elaborate on how to use human induced pluripotent stem cells for cardiomyocyte differentiation and purification, and further, on how to improve its transplantation efficiency with Rho-associated protein kinase inhibitor pretreatment in a mouse myocardial infarction model.

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. ...

Biochemistry

Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that mouse embryonic, dermal, and cardiac fibroblasts can be reprogrammed into functional induced-cardiomyocyte-like cells (iCMs) through overexpression of cardiogenic transcription factors including GATA4, Hand2, Mef2c, and Tbx5 both in vitro and in vivo. However, these previous studies have shown relatively low efficiency. In order to restore heart function following injury, mechanisms governing cardiac reprogramming must be elucidated to increase efficiency and maturation of iCMs.
We previously demonstrated that inhibition of pro-fibrotic signaling dramatically increases reprogramming efficiency. Here, we detail methods to achieve a reprogramming efficiency of up to 60%. Furthermore, we describe several methods including flow cytometry, immunofluorescent imaging, and calcium imaging to quantify reprogramming efficiency and maturation of reprogrammed fibroblasts. Using the protocol detailed here, mechanistic studies can be undertaken to determine positive and negative regulators of cardiac reprogramming. These studies may identify signaling pathways that can be targeted to promote reprogramming efficiency and maturation, which could lead to novel cell therapies to treat human heart disease.

Here we present a robust method to reprogram primary embryonic fibroblasts into functional cardiomyocytes through overexpression of GATA4, Hand2, Mef2c, Tbx5, miR-1, and miR-133 (GHMT2m) alongside inhibition of TGF-&#946; signaling. Our protocol generates beating cardiomyocytes as early as 7 days post-transduction with up to 60% efficiency.

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

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 ...

Biology

Murine Short Axis Ventricular Heart Slices for Electrophysiological Studies

Murine cardiomyocytes have been extensively used for in vitro studies of cardiac physiology and new therapeutic strategies. However, multicellular preparations of dissociated cardiomyocytes are not representative of the complex in vivo structure of cardiomyocytes, non-myocytes and extracellular matrix, which influences both mechanical and electrophysiological properties of the heart. Here we describe a technique to prepare viable ventricular slices of adult mouse hearts with a preserved in vivo like tissue structure, and demonstrate their suitability for electrophysiological recordings. After excision of the heart, ventricles are separated from the atria, perfused with Ca2+-free solution containing 2,3-butanedione monoxime and embedded in a 4% low-melt agarose block. The block is placed on a microtome with a vibrating blade, and tissue slices with a thickness of 150-400 &#181;m are prepared keeping the vibration frequency of the blade at 60-70 Hz and moving the blade forward as slowly as possible. Thickness of the slices depends on the further application. Slices are stored in ice cold Tyrode&#39;s solution with 0.9 mM Ca2+ and 2,3-butanedione monoxime (BDM) for 30 min. Afterwards, slices are transferred to 37 &#176;C DMEM for 30 min to wash out the BDM. Slices can be used for electrophysiological studies with sharp electrodes or micro electrode arrays, for force measurements to analyze contractile function or to investigate the interaction of transplanted stem cell-derived cardiomyocytes and host tissue. For sharp electrode recordings, a slice is placed into a 3 cm cell culture dish on the heating plate of an inverted microscope. The slice is stimulated with a unipolar electrode, and intracellular action potentials of cardiomyocytes within the slice are recorded with a sharp glass electrode.

Here, we describe the preparation of viable ventricular slices from adult mice and their use for sharp electrode action potential recordings. These multicellular preparations provide a preserved in vivo like tissue structure, which makes them a valuable model for electrophysiological and pharmacological studies in vitro.

Murine cardiomyocytes have been extensively used for in vitro studies of cardiac physiology and new therapeutic strategies. However, multicellular ...

Medicine

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 ...

Electromechanical Assessment of Optogenetically Modulated Cardiomyocyte Activity

Over the past two decades, optogenetic tools have been established as potent means to modulate cell-type specific activity in excitable tissues, including the heart. While Channelrhodopsin-2 (ChR2) is a common tool to depolarize the membrane potential in cardiomyocytes (CM), potentially eliciting action potentials (AP), an effective tool for reliable silencing of CM activity has been missing. It has been suggested to use anion channelrhodopsins (ACR) for optogenetic inhibition. Here, we describe a protocol to assess the effects of activating the natural ACR GtACR1 from Guillardia theta in cultured rabbit CM. Primary readouts are electrophysiological patch-clamp recordings and optical tracking of CM contractions, both performed while applying different patterns of light stimulation. The protocol includes CM isolation from rabbit heart, seeding and culturing of the cells for up to 4 days, transduction via adenovirus coding for the light-gated chloride channel, preparation of patch-clamp and carbon fiber setups, data collection and analysis. Using the patch-clamp technique in whole-cell configuration allows one to record light-activated currents (in voltage-clamp mode, V-clamp) and AP (current-clamp mode, I-clamp) in real time. In addition to patch-clamp experiments, we conduct contractility measurements for functional assessment of CM activity without disturbing the intracellular milieu. To do so, cells are mechanically preloaded using carbon fibers and contractions are recorded by tracking changes in sarcomere length and carbon fiber distance. Data analysis includes assessment of AP duration from I-clamp recordings, peak currents from V-clamp recordings and force calculation from carbon fiber measurements. The described protocol can be applied to the testing of biophysical effects of different optogenetic actuators on CM activity, a prerequisite for the development of a mechanistic understanding of optogenetic experiments in cardiac tissue and whole hearts.

We present a protocol for evaluating the electromechanical effects of GtACR1 activation in rabbit cardiomyocytes. We provide detailed information on cell isolation, culturing and adenoviral transduction, and on functional experiments with the patch-clamp and carbon-fiber techniques.

Over the past two decades, optogenetic tools have been established as potent means to modulate cell-type specific activity in excitable tissues, including ...

A Cardiac Microphysiological System for Studying Ca2+ Propagation via Non-genetic Optical Stimulation

In vitro cardiac microphysiological models are highly reliable for scientific research, drug development, and medical applications. Although widely accepted by the scientific community, these systems are still limited in longevity due to the absence of non-invasive stimulation techniques. Phototransducers provide an efficient stimulation method, offering a wireless approach with high temporal and spatial resolution while minimizing invasiveness in stimulation processes. In this manuscript, we present a fully optical method for stimulating and detecting the activity of an in vitro cardiac microphysiological model. Specifically, we fabricated engineered laminar anisotropic tissues by seeding human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) generated in a 3D bioreactor suspension culture. We employed a phototransducer, an amphiphilic azobenzene derivative, named Ziapin2, for stimulation and a Ca2+ dye (X-Rhod 1) for monitoring the system&#39;s response. The results demonstrate that Ziapin2 can photomodulate Ca2+ responses in the employed system without compromising tissue integrity, viability, or behavior. Furthermore, we showed that the light-based stimulation approach offers a similar resolution compared to electrical stimulation, the current gold standard. Overall, this protocol opens promising perspectives for the application of Ziapin2 and material-based photostimulation in cardiac research.

Using light to control cardiac cells and tissue enables non-contact stimulation, thereby preserving the natural state and function of the cells, making it a valuable approach for both basic research and therapeutic applications.

In vitro cardiac microphysiological models are highly reliable for scientific research, drug development, and medical applications. Although widely ...

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

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