This work was financially supported by the Kosair Charities Pediatric Heart Research Program at the University of Louisville and the Organoid Project at the RIKEN Center for Biosystems Dynamics Research. HiPSCs used in our published protocols were provided by the Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan.

1. Maintenance of hiPSCs and cardiovascular differentiation
<ol>
	<li>Expand and maintain hiPSCs on thin-coat basement membrane matrix (growth factor reduced, 1:60 dilution) in conditioned medium extracted from mouse embryonic fibroblasts (MEF-CM) with human basic fibroblast growth factor (hbFGF)4. 
	NOTE: We used a hiPSCs (4-factor (Oct3/4, Sox2, Klf4 and c-Myc) line: 201B6). Add hbFGF at the appropriate concentration for each cell line. Laminin-511 fragment can also be used for the coating of culture dish instead of basement membrane matrix. Commercially available medium designed for feeder-free culture of hiPSCs can be used as a substitute for MEF-CM.</li>
	<li>Use Versene (0.48 mM ethylenediaminetetraacetic acid (EDTA) solution) to detach and dissociate cells when cell confluency reaches 90&#8210;100%. 
	NOTE: Other commercially available products for cell dissociation can also be used.</li>
	<li>Plate cells at a density of 10,000 cells/mm2 on matrix-coated cell culture plates in MEF-CM with hbFGF and culture for two to three days.</li>
	<li>When the culture becomes fully confluent, cover the cells with matrix (1:60 dilution with MEF-CM) for one day.</li>
	<li>Replace the MEF-CM with RPMI 1640 + B27 medium. Add 100 ng/mL of Activin A and 100 ng/mL of Wnt3A to the medium for one day. 
	NOTE: This is day 0 of differentiation. To upregulate canonical Wnt signaling, GSK3&#946; inhibitors can also be used instead of Wnt3A.</li>
	<li>On day 1 of differentiation, change the medium to new RPMI 1640 + B27 with 10 ng/mL of BMP4 and 10 ng/mL of hbFGF. Culture cells for two (MC protocol) or four (CM+EC protocol) days without medium change5. 
	NOTE: CM+EC protocol is optimized to simultaneously induce cardiomyocytes (CMs) and vascular endothelial cells (ECs). MC protocol is optimized to preferentially induce vascular mural cells (MCs).</li>
	<li>CM+EC protocol for the induction of CMs and ECs (Figure 1A).
	<ol>
		<li>Replace the medium on day 5 of differentiation with RPMI 1640 + B27 supplemented with 50 ng/mL of VEGF165.</li>
		<li>Change the culture medium every 48 h until day 13&#8210;15 of differentiation.</li>
	</ol>
	</li>
	<li>MC protocol for the induction of vascular mural cells (Figure 1B)
	<ol>
		<li>Replace the medium with RPMI1640 + 10% fetal bovine serum (FBS) at day 3 of differentiation.</li>
		<li>Change the culture medium every 48 h until day 13&#8210;15 of differentiation.</li>
	</ol>
	</li>
</ol>
2. Cell harvest and lineage analysis on differentiation day 13&#8210;15
<ol>
	<li>Wash the cells with Ca2+ and Mg2+ free phosphate-buffered saline (PBS).</li>
	<li>Add cell dissociation solution (containing proteases, collagenases and DNAses) in the cell culture dish to cover the plate. Incubate the plate for 5 min at 37 &#176;C.</li>
	<li>Collect and dissociate the cells with culture medium using a pipette after incubation.</li>
	<li>Allocate 1 x 106 cells for lineage analysis with flow cytometry. To eliminate dead cells, stain the cells with fixable viability dye.</li>
	<li>Stain the cells with membrane surface markers in PBS with 5% FBS. Use the following dilutions of antibody in fluorescence activated cell sorting (FACS) staining buffer: anti-PDGFR&#946; (1:100), anti-VE cadherin (1:100), anti-TRA-1-60 (1:20).</li>
	<li>For intracellular proteins, resuspend and fix the cells with 4% paraformaldehyde (PFA) in PBS.</li>
	<li>Stain the cells with anti-cardiac isoform of Troponin T (cTnT) in PBS with 5% FBS and 0.75% Saponin, then label the cTnT antibody with 488 mouse IgG1 (dilution 1:50).</li>
	<li>Resuspend the stained cells in PBS with 5% FBS and put them in FACS tubes with cell strainer.</li>
	<li>Analyze the cell composition of the stained cells from each differentiation protocol with flow cytometry to facilitate the generation of cell suspensions with defined lineage distributions. 
	NOTE: While performing this procedure, the remaining cell suspension for ECTs is preserved in a 4 &#176;C refrigerator.</li>
</ol>
3. Fabrication of PDMS tissue mold
<ol>
	<li>Cast a 0.5 mm thick and over 30 mm x 30 mm layer of polydimethylsiloxane (PDMS) by mixing the prepolymer and cross-linking solution at a ratio of 10:1 and then cure at 80 &#176;C for 3 h.</li>
	<li>Cut the PDMS sheet and bond it with silicone adhesive to fabricate a 21 mm x 20.5 mm rectangular tray with 7 mm long, 0.5 mm wide, and 2.5 mm high rectangular posts at a staggered position. Horizontal post spacing between two lines of posts is 2.5 mm (Figure 2B).</li>
	<li>Autoclave the tray at 120 &#176;C for 20 min.</li>
	<li>Coat the tray with 1% poloxamer 407 in PBS for 1 h. Rinse the poloxamer 407 and rinse the mold with PBS sufficiently prior to use.</li>
</ol>
4. ECT construction
<ol>
	<li>After the cell lineage analysis, combine the cells from CM+EC and MC protocols so that the final concentration of MCs is 10 to 20% in a total cell number of six million cells per construct5.</li>
	<li>Suspend mixed six million cells in ECT culture medium (alpha minimum essential medium supplemented with 10% FBS, 50 &#181;M 2-mercaptoethanol and 100 U/mL Penicillin-Streptomycin).</li>
	<li>Preparation of matrix solution
	<ol>
		<li>Mix 133 &#181;L of acid-soluble rat tail collagen type I solution (2 mg/mL, pH 3) with 17 &#181;L of 10x minimum essential medium (MEM). Then mix the solution with 17 &#181;L of alkali buffer (0.2 M NaHCO3, 0.2 M HEPES, and 0.1 M NaOH). 
		NOTE: Collagen must be kept on ice (4 &#176;C). Check buffer color and if the medium does not become pinkish when all solutions are mixed, add additional alkali buffer to the medium. The mixing steps must be mix collagen I + 10x MEM, and then add alkali buffer. DO NOT change this order. Do NOT generate bubbles in the mix.</li>
		<li>Add 67 &#181;L of basement membrane matrix to the neutralized collagen solution. 
		NOTE: The mixed solution must be kept on ice (4 &#176;C).</li>
	</ol>
	</li>
	<li>Centrifuge the prepared cell suspension containing six million cells at 1,100 rpm (240 x g) for 5 min and resuspend the cells with 167 &#181;L of high-glucose DMEM + 20% FBS + 1% penicillin-streptomycin (100x).</li>
	<li>Mix the cell suspension and the matrix solution. The total volume of cell/matrix mixture for one construct is 400 &#956;L. 
	NOTE: The cell/matrix mixture should be non-viscous and a pinkish color at this step. Keep it on ice as the gel will solidify at room temperature. The final concentration of collagen type I is 0.67 mg/mL.</li>
	<li>Pour the cell/matrix mixture evenly over the poloxamer 407 coated PDMS tissue mold, which is placed in a six-well culture plate12. 
	NOTE: Pour the mixture carefully to avoid generating bubbles in order to prevent filling defects in the poured gel.</li>
	<li>Incubate the cell/matrix mixture in a standard CO2 incubator (37C, 5 % CO2) for 60 min.</li>
	<li>After the tissue is formed, soak the tissue mold with 4 mL of ECT culture medium. 
	NOTE: Although the cell/matrix is crosslinked in 60 min, the construct is still very fragile. Add medium gently to avoid damaging it.</li>
	<li>Culture the tissue for 14 days with medium change every day.</li>
	<li>Prior to ECT implantation, remove the ECT from the loading posts gently using sterilized fine forceps. 
	NOTE: The final ECT dimensions after removal from the mold are less than the original mold. A 2.1 cm x 2.05 cm mold generates a released ECT of approximately 1.5 cm x 1.5 cm. A larger 3.9 cm x 4.05 cm mold generates an ECT of approximately 3 cm x 3 cm. Unloaded ECT shows intrinsic spontaneous beating in warm culture medium. Although it initially maintains a mesh structure, an unloaded ECT shrinks and condenses over time. It is possible to hold the tissue softly with fine forceps and then use 7-0 silk suture to attach the ECT to the epicardium.</li>
</ol>

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A., Doree, C., Mathur, A., Martin-Rendon, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meta-Analysis+of+Cell+Therapy+Trials+for+Patients+With+Heart+Failure.">Meta-Analysis of Cell Therapy Trials for Patients With Heart Failure.</a> Circulation Research. 116, 1361-1377 (2015).</li><li>Menasch&#233;, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cell-derived+cardiac+progenitors+for+severe+heart+failure+treatment:+first+clinical+case+report:+Figure+1.">Human embryonic stem cell-derived cardiac progenitors for severe heart failure treatment: first clinical case report: Figure 1.</a> European Heart Journal. 36, 2011-2017 (2015).</li><li>Masumoto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+iPS+cell-engineered+cardiac+tissue+sheets+with+cardiomyocytes+and+vascular+cells+for+cardiac+regeneration.">Human iPS cell-engineered cardiac tissue sheets with cardiomyocytes and vascular cells for cardiac regeneration.</a> Scientific Reports. 4, 6716 (2014).</li><li>Masumoto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+myocardial+regenerative+potential+of+three-dimensional+engineered+cardiac+tissues+composed+of+multiple+human+iPS+cell-derived+cardiovascular+cell+lineages.">The myocardial regenerative potential of three-dimensional engineered cardiac tissues composed of multiple human iPS cell-derived cardiovascular cell lineages.</a> Scientific Reports. 6, 29933 (2016).</li><li>Zimmermann, W. 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The authors have no financial or scientific conflicts to disclose.

Following the completion of our investigation of a linear format, hiPSC derived ECT5, we adapted the protocol to mix hiPSC-derived CMs, ECs, and MCs to facilitate the in vitro expansion of vascular cells within ECTs and subsequent in vivo vascular coupling between ECTs and recipient myocardium.
To facilitate the generation of larger, implantable mesh ECT geometries we used thin PDMS sheets to design the 3D molds with loading posts arrayed at staggered positions. During ...

Numerous preclinical studies and clinical trials have confirmed the efficiency of cell-based cardiac regenerative therapies for failing hearts1,2,3. Among various cell types, human induced pluripotent stem cells (hiPSCs) are promising cell sources by virtue of their proliferative ability, potential to generate various cardiovascular lineages4,5, and allogenicity. In addition, tissue engineering technologies have made it possible to transfer millions of cells onto a damaged heart5,6,7,8.
Previously, we reported the generation of three-dimensional (3D) linear engineered cardiac tissues (ECTs) from hiPSC-derived cardiovascular lineages using a commercially available culture system for 3D bioartificial tissues5,7. We found that the coexistence of vascular endothelial cells and mural cells with cardiomyocytes within the ECT facilitated structural and electrophysiological tissue maturation. Furthermore, we validated the therapeutic potential of implanted hiPSC-ECTs in an immune tolerant rat myocardial infarction model to improve cardiac function, regenerate myocardium, and enhance angiogenesis5. However, the linear ECTs constructed by this method were 1 mm by 10 mm cylinders and therefore not suitable for the implantation in preclinical studies with larger animals or clinical use.
Based on the successful use of tissue molds to generate porous engineered tissue formation using rat skeletal myoblasts and cardiomyocytes9, human ESC-derived cardiomyocytes10 and mouse iPSCs11, we developed a protocol to generate scalable hiPSC-derived larger implantable tissue using polydimethylsiloxane (PDMS) molds. We evaluated a range of mold geometries to determine the most effective mold characteristics. Mesh-shaped ECTs with multiple bundles and junctions exhibited excellent characteristics in cell viability, tissue function and scalability compared to plain-sheet or linear formats that lacked pores or junctions. We implanted the mesh-shaped ECT in a rat myocardial infarction model and confirmed its therapeutic effects similar to implanted cylindrical ECTs12. Here we describe the protocol to generate a hiPSC-derived mesh-shaped ECT.

<table><tbody><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cell Culture Dishes 100x20 mm style</td><td>Falcon/ Thomas scientific</td><td>9380C51</td><td></td></tr><tr><td>Multiwell Plates For Cell Culture 6well 50/CS</td><td>Falcon / Thomas scientific</td><td>6902A01</td><td></td></tr><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Dow Corning</td><td>761036</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Accumax</td><td>Innovative Cell Technologies</td><td>AM-105</td><td></td></tr><tr><td>BMP4, recombinant (10&amp;micro;g)</td><td>R&amp;amp;D</td><td>RSD-314-BP-010</td><td></td></tr><tr><td>Collagen, Type I solution from rat tail</td><td>Sigma</td><td>C3867</td><td></td></tr><tr><td>Growth factor-reduced Matrigel</td><td>Corning</td><td>356231</td><td></td></tr><tr><td>Human VEGF (165) IS, premium grade</td><td>Miltenyi</td><td>130-109-385</td><td></td></tr><tr><td>Pluronic F-127, 0.2 &amp;micro;m filtered (10% Solution in Water)</td><td>Molecular Probes</td><td>P-6866</td><td></td></tr><tr><td>Recombinant human bFGF</td><td>WAKO</td><td>060-04543</td><td></td></tr><tr><td>Recombinant Human/Mouse/Rat ActivinA (50&amp;micro;g)</td><td>R&amp;amp;D</td><td>338-AC-050</td><td></td></tr><tr><td>rh Wnt-3a (10&amp;micro;g)</td><td>R&amp;amp;D</td><td>5036-WN</td><td></td></tr><tr><td>Versene solution</td><td>Gibco</td><td>15040066</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Culture medium and supplements&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10x MEM</td><td>Invitrogen</td><td>11430</td><td></td></tr><tr><td>2 Mercaptro Ethanol</td><td>SIGMA</td><td>M6250</td><td></td></tr><tr><td>B27 supplement minus insulin</td><td>Gibco</td><td>A1895601</td><td></td></tr><tr><td>DMEM, high glucose</td><td>Gibco</td><td>11965084</td><td></td></tr><tr><td>Fetal Bovine Serum (500ml)</td><td>Any</td><td></td><td></td></tr><tr><td>Fetal Bovine Serum (500ml)</td><td>Any</td><td></td><td></td></tr><tr><td>L-Glutamine</td><td>Gibco</td><td>25030081</td><td></td></tr><tr><td>NaHCO3</td><td>Any</td><td></td><td></td></tr><tr><td>PBS 1x</td><td>Gibco</td><td>10010-031</td><td></td></tr><tr><td>Penicillin-Streptomycin (5000 U/mL)</td><td>Gibco</td><td>15070-063</td><td></td></tr><tr><td>RPMI1640 medium</td><td>Gibco</td><td>21870092</td><td></td></tr><tr><td>&amp;alpha;MEM</td><td>Invitrogen</td><td>11900024</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Flowcytometry&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>anti-TRA-1-60, FITC, Clone: TRA-1-60, BD Biosciences</td><td>BD / Fisher</td><td>560380</td><td></td></tr><tr><td>anti-Troponin T, Cardiac Isoform Ab-1, Clone: 13-11, Thermo Scientific Lab Vision</td><td>Fisher</td><td>MS-295-P0</td><td></td></tr><tr><td>BD FACS Clean Solution</td><td>BD</td><td>340345</td><td></td></tr><tr><td>BD FACSFlow Sheath Fluid</td><td>BD</td><td>342003</td><td></td></tr><tr><td>BD FACSRinse Solution</td><td>BD</td><td>340346</td><td></td></tr><tr><td>EDTA</td><td>Any</td><td></td><td></td></tr><tr><td>Falcon Tube with Cell Strainer Cap (Case of 500)</td><td>Corning</td><td>352235</td><td></td></tr><tr><td>Fetal Bovine Serum (500ml)</td><td>Any</td><td></td><td></td></tr><tr><td>LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, for 405 nm excitation</td><td>Molecular Probes</td><td>L34957</td><td></td></tr><tr><td>PDGFRb; anti-CD140b, R-PE, Clone: 28D4, BD Biosciences</td><td>BD / Fisher</td><td>558821</td><td></td></tr><tr><td>Saponin</td><td>Sigma-Aldrich</td><td>47306-50G-F</td><td></td></tr><tr><td>VEcad-FITC; anti-CD144, FITC, Clone: 55-7H1, BD Biosciences</td><td>BD / Fisher</td><td>560411</td><td></td></tr><tr><td>Zenon Alexa Fluor 488 Mouse IgG1 Labeling Kit</td><td>Molecular Probes</td><td>Z25002</td><td></td></tr></tbody></table>

preparation of mesh-shaped engineered cardiac tissues derived from human ips cells for in vivo myocardial repair

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from human induced pluripotent stem cells (hiPSCs), which are developed towards the goal of clinical use. HiPSC-derived cardiomyocytes, endothelial cells, and vascular mural cells are mixed with gel matrix and then poured into a polydimethylsiloxane (PDMS) tissue mold with rectangular internal staggered posts. By culture day 14 ECTs mature into a 1.5 cm x 1.5 cm mesh structure with 0.5 mm diameter myofiber bundles. Cardiomyocytes align to the long-axis of each bundle and spontaneously beat synchronously. This approach can be scaled up to a larger (3.0 cm x 3.0 cm) mesh ECT while preserving construct maturation and function. Thus, mesh-shaped ECTs generated from hiPSC-derived cardiac cells may be feasible for cardiac regeneration paradigms.

Figure 1A,B shows the schematics of CM+EC and MC protocol. After inducing CMs and ECs from CM+EC protocol and MCs from MC protocol, the cells are mixed adjusting final MC concentrations to represent 10 to 20% of total cells. The 2 cm&#160;wide tissue mold is fabricated according to the design drawing from 0.5 mm thick PDMS sheet (Figure 2A,B). Six million of CM+EC+MC cells are combined with collagen I, and matrix and poured onto...

Watch this Scientific Journal Video about Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair at JoVE.com

Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair

The present protocol generates mesh-shaped engineered cardiac tissues containing cardiovascular cells derived from human induced pluripotent stem cells to allow the investigation of cell implantation therapy for heart diseases.

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from ...

preparation-mesh-shaped-engineered-cardiac-tissues-derived-from-human

Research

JoVE Journal

Bioengineering

5.4K Views.  RIKEN Center for Biosystems Dynamics Research. Dit protocol beschrijft een nobele en eenvoudige methode voor het creëren van mesh-vormige ontworpen cardiale weefsels afgeleid van menselijke geïnduceerde lichaamsdelen en STAMcel afgeleide hartcellen. De flexibiliteit van de weefselgeometrie en de schaalbaarheid van de bewaarde weefselfunctie zijn de belangrijkste voordelen van deze techniek. De geïmplanteerde mesh vorm ECT's kan herstellen van een cardiale structuur en functie in een rat hartinfarct model.Bevestiging van de haalb...

Video: Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing

Advanced heart failure represents a major unmet clinical challenge, arising from the loss of viable and/or fully functional cardiac muscle cells. Despite optimum drug therapy, heart failure represents a leading cause of mortality and morbidity in the developed world. A major challenge in drug development is the identification of cellular assays that accurately recapitulate normal and diseased human myocardial physiology in vitro. Likewise, the major challenges in regenerative cardiac biology revolve around the identification and isolation of patient-specific cardiac progenitors in clinically relevant quantities. These cells have to then be assembled into functional tissue that resembles the native heart tissue architecture. Microcontact printing allows for the creation of precise micropatterned protein shapes that resemble structural organization of the heart, thus providing geometric cues to control cell adhesion spatially. Herein we describe our approach for the isolation of highly purified myocardial cells from pluripotent stem cells differentiating in vitro, the generation of cell growth surfaces micropatterned with extracellular matrix proteins, and the assembly of the stem cell-derived cardiac muscle cells into anisotropic myocardial tissue.

The generation of aligned myocardial tissue is a key requirement for adapting the recent advances in stem cell biology to clinically useful purposes. Herein we describe a microcontact printing approach for the precise control of cell shape and function. Using highly purified populations of embryonic stem cell derived cardiac progenitors, we then generate anisotropic functional myocardial tissue.

Advanced heart failure represents a major unmet clinical challenge,  arising from the loss of viable and/or fully functional cardiac muscle  cells. ...

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 ...

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 ...

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. Human-induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs), human cardiac fibroblasts (HCFs), and human umbilical vein endothelial cells (HUVECs) are isolated and used to generate a cell suspension with 70% iPSC-CMs, 15% HCFs, and 15% HUVECs. They are co-cultured in an ultra-low attachment "hanging drop" system, which contains micropores for condensing hundreds of spheroids at one time. The cells aggregate and spontaneously form beating spheroids after 3 days of co-culture. The spheroids are harvested, seeded into a novel mold cavity, and cultured on a shaker in the incubator. The spheroids become a mature functional tissue approximately 7 days after seeding. The resultant multilayered tissues consist of fused spheroids with satisfactory structural integrity and synchronous beating behavior. This new method has promising potential as a reproducible and cost-effective method to create engineered tissues for the treatment of heart failure in the future.

This protocol describes a net mold-based method to create three-dimensional scaffold-free cardiac tissues with satisfactory structural integrity and synchronous beating behavior.

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...

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 ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

Presented here is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human-induced pluripotent stem cell-derived cardiomyocytes cultured in a high-throughput, multidimensional format. This three-dimensional model functions as a robust platform for disease modeling, high-throughput screenings, and maintains its functionality after cryopreservation.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We ...

Designing a Bioreactor to Improve Data Acquisition and Model Throughput of Engineered Cardiac Tissues

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering is crucial for basic science cardiac research; in vitro human cell culture eliminates the interspecies differences of animal models, while a more tissue-like 3D environment (e.g., with extracellular matrix and heterocellular coupling) simulates in vivo conditions to a greater extent than traditional two-dimensional culture on plastic Petri dishes. However, each model system requires specialized equipment, for example, custom-designed bioreactors and functional assessment devices. Additionally, these protocols are often complicated, labor-intensive, and plagued by the failure of the small, delicate tissues.
This paper describes a process for generating a robust human engineered cardiac tissue (hECT) model system using induced pluripotent stem-cell-derived cardiomyocytes for the longitudinal measurement of tissue function. Six hECTs with linear strip geometry are cultured in parallel, with each hECT&#160;suspended from a pair of force-sensing polydimethylsiloxane (PDMS) posts attached to PDMS racks. Each post is capped with a black PDMS stable post tracker (SPoT), a new feature that improves the ease of use, throughput, tissue retention, and data quality. The shape allows for the reliable optical tracking of post deflections, yielding improved twitch force tracings with absolute active and passive tension. The cap geometry eliminates tissue failure due to hECTs slipping off the posts, and as they involve a second step after PDMS rack fabrication, the SPoTs can be added to existing PDMS post-based designs without major changes to the bioreactor fabrication process.
The system is used to demonstrate the importance of measuring hECT function at physiological temperatures and shows stable tissue function during data acquisition. In summary, we describe a state-of-the-art model system that reproduces key physiological conditions to advance the biofidelity, efficiency, and rigor of engineered cardiac tissues for in vitro applications.

Three-dimensional cardiac tissues bioengineered using stem-cell-derived cardiomyocytes have emerged as promising models for studying healthy and diseased human myocardium in vitro while recapitulating key aspects of the native cardiac niche. This manuscript describes a protocol for fabricating and analyzing high-content engineered cardiac tissues generated from human induced pluripotent stem-cell-derived cardiomyocytes.

Heart failure remains the leading cause of death worldwide, creating a pressing need for better preclinical models of the human heart. Tissue engineering ...

Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair

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