This work was supported by a grant-in-aid from the Heart and Stroke Foundation of Canada (G-14-0006265), operating grants from the Canadian Institutes of Health Research (137352 and 143066), and a J.P. Bickell foundation grant (1013821) to SSN.

1. Master Design and Fabrication
NOTE: Use soft lithography for device fabrication. Make a two-layer SU-8 master for polydimethylsiloxane (PDMS) molding.
<ol>
	<li>Design the device using a design and drafting software (Figure 1A, left). Draw each layer of the master separately. Print the device design on two photomasks at 20,000 dpi, corresponding to the two layers of the master15. Set the device pattern as transparent and the surrounding area as dark; the masks will serve as the template to optically transfer the device pattern onto SU-8 by lithography (as described by others15).</li>
	<li>Dispense about 4 mL of SU-8 50 onto the center of a 4 inch silicon wafer. Coat the SU-8 50 evenly on the wafer using a spin coater by spinning at 2,000 revolutions per minute (RPM) for 30 s. Bake the wafer on a 95 &#176;C hotplate for 10 min. Expose the wafer to ultraviolet (UV) light at 200 mJ/cm2.</li>
	<li>Pour about 4 mL of SU-8 2050 onto the center of the wafer and spin at 2,500 RPM for 30 s. Bake on the 95 &#176;C hotplate for 15 min. Cool to room temperature (RT).</li>
	<li>Repeat step 1.3 two more times.</li>
	<li>Cover the coated wafer with the first-layer transparency photomask. Expose the wafer to UV light at 270 mJ/cm2 to make the first layer. Bake on the 95 &#176;C hotplate for 15 min.</li>
	<li>Make the second layer of SU-8 2050 by repeating step 1.3 twice.</li>
	<li>Align the second-layer mask to the features on the first layer using a mask aligner. Then, expose to UV light at 240 mJ/cm2. Bake at 95 &#176;C for 15 min. 
	NOTE: The exposure time is determined by the required total UV dose and the UV light output intensity measured on the day prior to use.</li>
	<li>Develop the wafer by submerging it in propylene glycol monomethyl ether acetate (PGMEA) and shaking at 200 RPM for 30 min on an orbital shaker.</li>
	<li>Place the master into a Petri dish and carefully pour the PDMS mixture (base to crosslinker at a ratio of 10:1 by weight) on the center of the wafer. Cover all the features and avoid air bubbles.</li>
	<li>Heat in an oven at 70 &#176;C for 2 h. Use a sharp blade to cut around the edge of the PDMS biowire template (Figure 1A, right). Peel the PDMS from the master. Trim the device with the blade. Put the PDMS biowire template in a sterilization pouch and steam autoclave at 121 &#176;C for 20 min.</li>
	<li>In a biosafety cabinet (BSC), place the PDMS biowire template into a Petri dish. Use tweezers to hold and mount a piece of sterile surgical silk suture (6-0) in the center of the channel of the PDMS microwell by seating it in the grooves at both ends of the channel (Figure 1B-I).</li>
</ol>
2. Creation of the Electrical Stimulation Chamber
<ol>
	<li>Make a pair of rectangular polycarbonate pieces (length x width x thickness: 2 cm x 0.85 cm x 0.35 cm) and use them as the frame of the electrical stimulation chamber (Figure 2).</li>
	<li>Drill two 3 mm wide holes 2 cm apart along the center line of each polycarbonate frame piece.</li>
	<li>Cut two pieces of 1.5 cm long carbon rods. Drill a 1 mm-wide hole through the carbon rod about 3 mm from the end. Thread a platinum wire through the carbon rod hole and tighten the wire by wrapping it around the carbon rod. Attach a clip to the other end of the platinum wire for later connection with the electric stimulator.</li>
	<li>Insert the carbon rods into the polycarbonate frame pieces. Place the assembled frame with the carbon rods into a well of a six-well plate.</li>
	<li>Pour 2 mL of PDMS into the well. Submerge the bottom of the polycarbonate frames in PDMS while keeping the PDMS surface level close to but not reaching the bottom of the carbon rods. Heat in an oven at 70 &#176;C for 2 h.</li>
	<li>Take the electrical stimulation chamber out of the six-well plate and put it in a sterilization pouch for steam sterilization at 121 &#176;C for 20 min.</li>
</ol>
3. Enzymatic Dissociation of hPSC-CMs
<ol>
	<li>Induce hPSC to differentiate into cardiomyocytes using an established protocol4. 
	NOTE: Briefly, generate cardiomyocytes via established embryoid body (EB) protocols in stem pro medium through the sequential supplementation with bone morphogenetic protein 4, basic fibroblast growth factor, activin A, vascular endothelial growth factor, and Dickkopf homolog 14. Use EBs from day 20-34 of differentiation to make biowires.</li>
	<li>Collect the EBs from the low attachment plate with a P1,000 pipette tip and transfer to a conical tube. Pellet by centrifugation for 5 min at 125 x g and RT. Discard the supernatant and resuspend the pellet with pre-warmed, 37 &#176;C collagenase type I containing 1% deoxyribonuclease I (DNAse I). Incubate at 37 &#176;C for 2 h for digestion.</li>
	<li>Add 5 mL of pre-warmed, 37 &#176;C wash medium and centrifuge for 5 min at 125 x g and RT. Discard the supernatant. Resuspend the pellet with 2 mL of trypsin/ethylenediaminetetraacetic acid (EDTA) solution and incubate at 37 &#176;C for 5 min.</li>
	<li>Add 1 mL of stop medium containing 3% (v/v) DNase I.</li>
	<li>Attach a 20 G (0.9 mm) needle to a 5 mL syringe and pass the EB suspension through it 3-5 times.</li>
	<li>Add 7 mL of wash medium and centrifuge at 280 x g for 5 min. Discard the supernatant. Resuspend the pellet in Iscove&#39;s modified Dulbecco&#39;s medium (IMDM) and store on ice. Determine the cell number with a hemocytometer. Take 0.5 x 106 cells and pellet by centrifugation at 280 x g for 5 min. Remove the supernatant.</li>
	<li>Pre-cool all the gel components on ice before mixing them in a sterile tube. Mix the following collagen gel components (final concentration): 2.1 mg/mL rat tail collagen type I, 24.9 mM glucose, 23.8 mM NaHCO3, 14.3 mM NaOH, 10 mM HEPES, and 10% extracellular matrix in 1x M199 medium. Add the collagen and extracellular matrix last. Mix by pipetting up and down.</li>
	<li>Resuspend 0.5 x 106 cells with 3.5 &#181;L of collagen gel and mix well.</li>
	<li>Use a P10 pipette tip to add 4 &#181;L of the cell suspension into the 0.5 cm-long channel (Figure 1B-II). Adjust the position of the suture with sterile forceps if necessary.</li>
	<li>Cover the bottom of the Petri dish with sterile phosphate-buffered saline (PBS), making sure not to touch the gel. Incubate at 37 &#176;C for 30 min.</li>
	<li>Replace the PBS with 12 mL of culture medium (e.g., stem pro or medium recommended by the cell manufacturer), enough to cover the cells (Figure 1B-III). Culture in an incubator at 37 &#176;C for 1 week. Change the medium every other day.</li>
</ol>
4. Electrical Stimulation for Biowire Cultivation
<ol>
	<li>In the BSC, add 2 mL of PBS to each well of a six-well plate that will house an electrical stimulator chamber. Use sterile tweezers to gently place the autoclaved electrical stimulation device into the well. Discard the PBS from the well.</li>
	<li>Cover the carbon rods with 5 mL of pre-warmed culture medium. Use sterile tweezers to mount the biowires in the electrical stimulation chamber and orient them perpendicular to the carbon rods (Figure 2).</li>
	<li>Place the lid on top of the six-well plate, but leave sufficient platinum wires reaching outside the plate to connect with the electrical stimulator. Place the six-well plate in a cell culture incubator at 37 &#176;C and connect the platinum wire to the electrical stimulator.</li>
	<li>Stimulate the biowires using an electrical stimulator with the following setting: biphasic repeating pulse, 1-ms pulse duration, 3 V/cm, and 1 pulse per second (PPS). Raise the PPS every 24 h to the following values: 1.83, 2.66, 3.49, 4.82, 5.15, and 6. Change the medium every other day.</li>
	<li>Observe the cardiomyocyte contractility in response to electrical stimulation under a microscope at 10X magnification. 
	NOTE: After one week of stimulation, the biowires becomes mature (see the results section).</li>
</ol>

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The authors declare that they have no competing financial interests.

This manuscript describes the setup and implementation of the engineered platform, biowire, to improve the maturation of hPSC-CMs. The device can be made in standard microfabrication facilities, and biowires can be produced with common cell culture techniques and an electrical stimulator.
To our knowledge, there is no reported method similar to biowires. This strategy reveals that improved maturation properties were dependent on the electrical stimulation regimen, as evidenced by the greater m...

Cell-based therapy is one of the most promising and investigated strategies to achieve cardiac repair/regeneration. It has been aided by cardiac tissue engineering and the co-delivery of biomaterials1,2. Most available cell sources have been studied in animal models for their potentially beneficial effects on damaged, diseased, or aged hearts3. In particular, significant efforts have been made to use human pluripotent stem cell (hPSC)-derived cardiomyocytes (hPSC-CM), a potentially unlimited autologous cell source for cardiac tissue engineering. hPSC-CMs can be produced using several established protocols4,5,6. However, the obtained cells display fetal-like phenotypes, with a range of immature characteristics compared to adult ventricular cardiomyocytes7,8. This can be an obstacle to the application of hPSC-CMs as models of adult heart tissue in drug discovery research and in the development of adult cardiac disease models9.
In order to overcome this limitation of phenotypic immaturity, new approaches have been actively investigated to promote cardiomyocyte maturation. Early studies revealed effective pro-maturation properties in neonatal rat cardiomyocytes via cyclic mechanical10 or electrical stimulation11. Gel compaction and cyclic mechanical stimulation were also shown to improve some aspects of hPSC-CM maturation12,13, with minimal enhancement of the electrophysiological and calcium handling properties. Therefore, a platform system called &#34;biological wire&#34; (biowire) was devised by providing both structural cues and electrical field stimulation to enhance the maturation of hPSC-CMs14. This system uses a microfabricated platform to create aligned cardiac tissue that is amenable to electrical field stimulation. This can be used to improve the structural and electrophysiological maturity of hPSC-CMs. Here, we describe the details of making such biowires.

<table><tbody><tr><td>L-Ascorbic acid</td><td>Sigma</td><td>A-4544</td><td>hPSC-CM culture media componet</td></tr><tr><td>AutoCAD</td><td>Autodesk, Inc</td><td></td><td>Software to design device</td></tr><tr><td>Carbon rods, &amp;Oslash; 3 mm</td><td></td><td></td><td>Electrical stimulator chamber component</td></tr><tr><td>Collagen, type 1, rat tail</td><td>BD Biosciences</td><td>354249</td><td>Collagen gel: 2.1 mg/mL of rat tail collagen type I in 24.9 mM glucose, 23.8 mM NaHCO&lt;sub&gt;3&lt;/sub&gt;, 14.3 mM NaOH, 10 mM HEPES, in 1x M199 media with 10 % of growth factor-reduced Matrigel.</td></tr><tr><td>Collagenase type I&amp;nbsp;</td><td>Sigma</td><td>C0130</td><td>0.2% collagenase type I (w/v) and 20% FBS (v/v) in PBS with Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;. Sterilize with 0.22 &amp;mu;m filter and make 12 mL aliquots. Store at -20 &amp;deg;C.</td></tr><tr><td>Deoxyribonuclease I (DNase I)</td><td>EMD Millipore</td><td>260913-25MU</td><td>Make 1 mg/mL DNase I stock solution in water. Filter sterile and store 0.5 mL aliquots at &amp;minus;20 &amp;deg;C</td></tr><tr><td>Drill &amp;amp; drill bits (&amp;Oslash; 1 mm and 2 mm)</td><td>Dremel</td><td></td><td>Drill holes in polycarbonate frames</td></tr><tr><td>Electrical stimulator</td><td>Grass</td><td>s88x</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>WISENT Inc.</td><td>080-450</td><td></td></tr><tr><td>D-(+)-Glucose&amp;nbsp;</td><td>Sigma</td><td>G5767</td><td>Collagen gel component</td></tr><tr><td>L-Glutamine</td><td>Thermo Fisher Scientific</td><td>25030081</td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O</td><td>MilliQ</td><td></td><td>18.2 M&amp;Omega;&amp;middot;cm at 25 &amp;deg;C, ultrapure, to make all solutions</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H4034</td><td>Collagen gel component</td></tr><tr><td>Hot plate</td><td>Torrey Pines</td><td>HS40</td><td></td></tr><tr><td>Iscove&#039;s Modified Dulbecco&#039;s Medium(IMDM)</td><td>Thermo Fisher Scientific</td><td>12440053</td><td></td></tr><tr><td>Mask aligner</td><td>EVG&amp;nbsp;</td><td>EVG 620</td><td></td></tr><tr><td>Matrigel, growth factor reduced&amp;nbsp;</td><td>Corning</td><td>354230</td><td>Collagen gel component</td></tr><tr><td>Medium 199 (M199)</td><td>Thermo Fisher Scientific</td><td>11825015</td><td>Collagen gel component</td></tr><tr><td>Monothioglycerol (MTG)</td><td>Sigma</td><td>M-6145</td><td>hPSC-CM culture media componet</td></tr><tr><td>Orbital shaker</td><td>VWR</td><td>89032-088</td><td></td></tr><tr><td>Penicillin/Streptomycin (P/S)</td><td>Thermo Fisher Scientific</td><td>15070063</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS) with Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>14040133</td><td></td></tr><tr><td>Plate (6-well)</td><td>Corning</td><td>353046</td><td></td></tr><tr><td>Plate (6-well), low attachment</td><td>Corning</td><td>3471</td><td></td></tr><tr><td>Platinum wires, 0.2 mm</td><td></td><td></td><td>Electrical stimulator chamber component</td></tr><tr><td>Polydimethylsiloxane (PDMS)</td><td>Dow Corning</td><td>Sylgard 184</td><td></td></tr><tr><td>Propylene glycol monomethyl ether acetate (PGMEA)</td><td>Doe &amp;amp; Ingalls Inc.</td><td></td><td>To develop the wafer</td></tr><tr><td>Pouch, peel-open</td><td>Convertors</td><td>92308</td><td>For steam sterilization</td></tr><tr><td>Silicon wafer, 4 inch</td><td>UniversityWafer Inc.</td><td></td><td></td></tr><tr><td>Sodium bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma</td><td>S5761</td><td>Collagen gel component</td></tr><tr><td>Sodium hydroxide</td><td>Sigma</td><td>S8045</td><td>Collagen gel component</td></tr><tr><td>Sprin coater</td><td>Specialty Coating Systems</td><td>G3P-8</td><td></td></tr><tr><td>StemPro-34 culture medium</td><td>Thermo Fisher Scientific</td><td>10639011</td><td>hPSC-CM culture medium. To make 50 mL, add 1.3 mL supplement, 500 &amp;mu;L of 100&amp;times; L-Glutamine, 250 &amp;mu;L of 30 mg/mL transferrin, 500 &amp;mu;l of 5 mg/mL ascorbic acid, 150 &amp;mu;L of 26 &amp;mu;l /2 mL monothioglycerol (MTG), and 500 &amp;mu;L (1 %) penicillin/streptomycin.</td></tr><tr><td>Stop media</td><td></td><td></td><td>Wash medium:FBS (1:1)</td></tr><tr><td>SU-8 50&amp;nbsp;</td><td>MicroChem Corp.</td><td></td><td>photoresist, master component</td></tr><tr><td>SU-8 2050&amp;nbsp;</td><td>MicroChem Corp.</td><td></td><td>photoresist, master component</td></tr><tr><td>Transferrin</td><td>Roche</td><td>10-652-202</td><td>hPSC-CM culture media componet</td></tr><tr><td>Trypsin/EDTA, 0.25%</td><td>Thermo Fisher Scientific</td><td>25200056</td><td>hPSC-CM culture media componet</td></tr><tr><td>Wash medium</td><td></td><td></td><td>IMDM containing 1% Penicillin/Streptomycin</td></tr></tbody></table>

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 rational for the use of a suture in the biowires is to serve as a template for the formation of 3D constructs that align in one axis and mimic the shape of cardiac fibers. We show that after seven days of culture in the biowire, cells remodeled the gel around the suture (Figure 3A). The cells assembled along the axis of the suture to form aligned cardiac tissue (Figure 3). After 7 days of preculture, the biowires were subjected to 7 days of electrical...

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

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 ...

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9.1K Views.  University Health Network.  De cardiale biowire platform is een in vitro werkwijze toegepast om menselijke embryonale en geïnduceerde pluripotente stamcellen afgeleide cardiomyocyten (HPSC-CM) rijpen combineert driedimensionale celkweek elektrische stimulatie. Dit handschrift bevat de gedetailleerde opzet van de cardiale biowire platform. 

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

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes (hPSC-CMs) Using Multi-electrode Arrays (MEAs)

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived cardiomyocytes (hPSC-CMs) are increasingly recognized as having great value for modeling cardiovascular diseases in humans, especially arrhythmia syndromes. They have also demonstrated relevance as in vitro systems for predicting drug responses, which makes them potentially useful for drug-screening and discovery, safety pharmacology and perhaps eventually for personalized medicine. This would be facilitated by deriving hPSC-CMs from patients or susceptible individuals as hiPSCs. For all applications, however, precise measurement and analysis of hPSC-CM electrical properties are essential for identifying changes due to cardiac ion channel mutations and/or drugs that target ion channels and can cause sudden cardiac death. Compared with manual patch-clamp, multi-electrode array (MEA) devices offer the advantage of allowing medium- to high-throughput recordings. This protocol describes how to dissociate 2D cell cultures of hPSC-CMs to small aggregates and single cells and plate them on MEAs to record their spontaneous electrical activity as field potential. Methods for analyzing the recorded data to extract specific parameters, such as the QT and the RR intervals, are also described here. Changes in these parameters would be expected in hPSC-CMs carrying mutations responsible for cardiac arrhythmias and following addition of specific drugs, allowing detection of those that carry a cardiotoxic risk.

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes hPSC-CMs Using Multi-electrode Arrays MEAs

Electrophysiological characterization of cardiomyocytes derived from human Pluripotent Stem Cells (hPSC-CMs) is crucial for cardiac disease modeling and for determining drug responses. This protocol provides the necessary information to dissociate and plate hPSC-CMs on multi-electrode arrays, measure their field potential, and a method for analyzing QT and RR intervals.

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

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.

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 ...

Bioengineering

Human iPSC-Derived Cardiomyocyte Networks on Multiwell Micro-electrode Arrays for Recurrent Action Potential Recordings

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput electrophysiological approaches for hiPSC-derived cardiomyocyte (hiPSC-CM) preparations is much needed for efficient drug testing. Although multielectrode arrays (MEAs) are frequently employed for field potential measurements of excitable cells, a recent publication by Joshi-Mukherjee and colleagues described and validated its application for recurrent action potential (AP) recordings from the same hiPSC-CM preparation over days. The aim here is to provide detailed step-by-step methods for seeding CMs and for measuring AP waveforms via electroporation with high precision and a temporal resolution of 1 &#181;s. This approach addresses the lack of easy-to-use methodology to gain intracellular access for high-throughput AP measurements for reliable electrophysiological investigations. A detailed work flow and methods for plating of hiPSC-CMs on multiwell MEA plates are discussed emphasizing critical steps wherever relevant. In addition, a custom-built MATLAB script for rapid data handling, extraction and analysis is reported for comprehensive investigation of the waveform analysis to quantify subtle differences in morphology for various AP duration parameters implicated in arrhythmia and cardiotoxicity.

This article contains a set of protocols for the development of human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) networks cultured on multiwell MEA plates to reversibly electroporate the cell membrane for action potential measurements. High-throughput recordings are obtained from the same cell sites repeatedly over days.

Cardiac safety screening is of paramount importance for drug discovery and therapeutics. Therefore, the development of novel high-throughput ...

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

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

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

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

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

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 ...

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

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

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

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

3D Human Myocardial Tissue Generation Using Melt Electrospinning Writing of Polycaprolactone Scaffolds and hiPSC-Derived Cardiac Cells

The development of functional human cardiac tissues holds significant promise for advancing applications in drug screening, disease modeling, and regenerative medicine. This protocol describes the stepwise fabrication of 3D myocardial tissues with advanced mimicry of native cardiac structure by combining melt electrospinning writing (MEW) polycaprolactone (PCL) scaffolds with fibrin hydrogels and human induced pluripotent stem cell (hiPSC)-derived cardiac cells. The process involves embedding a mixture of cardiomyocytes (hiPSC-CMs) and cardiac fibroblasts (hiPSC-CFs) within a fibrin matrix to create mini-tissues, with structural support provided by MEW-generated scaffolds. These fibrillar scaffolds are fabricated at the micro- to nanoscale, allowing for precise control over fiber architecture, which plays a key role in organizing cell distribution and alignment. Meanwhile, the fibrin matrix promotes cell viability and mimics the extracellular environment. Characterization of the generated tissues reveals well-organized sarcomeres within hiPSC-CMs, along with stable contractile activity. The tissues demonstrate consistent spontaneous beating as early as two days post-seeding, with sustained functionality over time. The combination of hiPSC-CFs with hiPSC-CMs enhances the structural integrity of the tissues while supporting long-term cell viability. This approach offers a reproducible, adaptable, and scalable method for creating biomimetic cardiac tissue models, providing a versatile platform for preclinical drug testing, mechanistic studies of cardiac disease, and potential regenerative therapies.

A reproducible method is presented to generate 3D myocardial tissues combining melt electrospinning writing (MEW) polycaprolactone (PCL) scaffolds and fibrin hydrogels with hiPSC-derived cardiomyocytes and fibroblasts. This technique offers precise control over scaffold architecture and can be applied in preclinical drug testing and cardiac disease modeling.

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

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