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 coatin...

<ol>
	<li>Sanganalmath, S. K., Bolli, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+therapy+for+heart+failure:+A+comprehensive+overview+of+experimental+and+clinical+studies,+current+challenges,+and+future+directions.">Cell therapy for heart failure: A comprehensive overview of experimental and clinical studies, current challenges, and future directions.</a> Circulation Research. 113, 810-834 (2013).</li><li>Fisher, S. 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. 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=Engineered+heart+tissue+grafts+improve+systolic+and+diastolic+function+in+infarcted+rat+hearts.">Engineered heart tissue grafts improve systolic and diastolic function in infarcted rat hearts.</a> Nature Medicine. 12, 452-458 (2006).</li><li>Fujimoto, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+fetal+cardiac+graft+preserves+its+cardiomyocyte+proliferation+within+postinfarcted+myocardium+and+sustains+cardiac+function.">Engineered fetal cardiac graft preserves its cardiomyocyte proliferation within postinfarcted myocardium and sustains cardiac function.</a> Tissue engineering. Part A. 17, 585-596 (2011).</li><li>Lancaster, J. J., 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=Surgical+treatment+for+heart+failure:+cell-based+therapy+with+engineered+tissue.">Surgical treatment for heart failure: cell-based therapy with engineered tissue.</a> Vessel Plus. 2019, (2019).</li><li>Bian, W., Liau, B., Badie, N., Bursac, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesoscopic+hydrogel+molding+to+control+the+3D+geometry+of+bioartificial+muscle+tissues.">Mesoscopic hydrogel molding to control the 3D geometry of bioartificial muscle tissues.</a> Nature protocols. 4, 1522-1534 (2009).</li><li>Zhang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+cardiac+patch+for+advanced+functional+maturation+of+human+ESC-derived+cardiomyocytes.">Tissue-engineered cardiac patch for advanced functional maturation of human ESC-derived cardiomyocytes.</a> Biomaterials. 34, 5813-5820 (2013).</li><li>Christoforou, N., 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=Induced+pluripotent+stem+cell-derived+cardiac+progenitors+differentiate+to+cardiomyocytes+and+form+biosynthetic+tissues.">Induced pluripotent stem cell-derived cardiac progenitors differentiate to cardiomyocytes and form biosynthetic tissues.</a> PloS one. 8, 65963 (2013).</li><li>Nakane, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+Cell+Composition+and+Geometry+on+Human+Induced+Pluripotent+Stem+Cells-Derived+Engineered+Cardiac+Tissue.">Impact of Cell Composition and Geometry on Human Induced Pluripotent Stem Cells-Derived Engineered Cardiac Tissue.</a> Scientific Reports. 7, 45641 (2017).</li><li>Kowalski, W. J., 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=Quantification+of+Cardiomyocyte+Alignment+from+Three-Dimensional+(3D)+Confocal+Microscopy+of+Engineered+Tissue.">Quantification of Cardiomyocyte Alignment from Three-Dimensional (3D) Confocal Microscopy of Engineered Tissue.</a> Microscopy and Microanalysis. 1, (2017).</li></ol>

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>Materials</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>Reagents</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&#181;g)</td>
			<td>R&#38;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 &#181;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&#181;g)</td>
			<td>R&#38;D</td>
			<td>338-AC-050</td>
			<td></td>
		</tr>
		<tr>
			<td>rh Wnt-3a (10&#181;g)</td>
			<td>R&#38;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>Culture medium and supplements</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>&#945;MEM</td>
			<td>Invitrogen</td>
			<td>11900024</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flowcytometry</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 ...

This protocol describes a noble and easy method for creating mesh-shaped engineered cardiac tissues derived from human induced body parts and STEM cell derived cardiac cells. The flexibility of the tissue geometry and the scalability of the preserved tissue function are the main advantages of this technique. The implanted mesh shape ECTs can restore a cardiac structure and function in a rat myocardial infarction model.Confirming its feasibility for use in cardiac regenerative therapy. These techniques require reproducible human STEM cell processing and the tissue culture skills for consistent results. Begin by cutting a cured PDMS sheet to the appropriate size to allow the sheet to be bonded with silicone adhesive to fabricate a 21 by 20.5 millimeter rectangular tray with seven millimeter long 0.5 millimeter wide and 2.5 millimeter high rectangular posts at staggered positions.The horizontal spacing between two lines of posts should be 2.5 millimeters. Autoclave the tray at 120 degrees Celsius for 20 minutes before coating the mold with 1%Poloxamer 407 in PBS for one hour. Then thoroughly rinse the mold with PBS and place the mold into one well of a six-well plate.For lineage analysis, combine the cells from the cardiomyocyte plus endothelial and vascular mural cell protocols. So that the final concentration of mural cells is 10 to 20%in a total cell number of six times 10 to the six cells per construct. Mix 133 microliters of Acid Soluble Rat Tail collagen type one solution and 17 microliters of Alkali Buffer to 17 microliters of 10X Minimum Essential Medium on ice.Then add 67 microliters of Basement Membrane Matrix to the collagen solution. Next, centrifuge the cells and resuspend the pellet in 167 microliters of High Glucose Dulbecco's Minimal Essential Medium supplemented with fetal bovine serum and antibiotics. Mix the cells on the matrix solution on ice.Then carefully pour 400 microliters of the cell matrix suspension onto the poloxamer 407 coated PDMS tissue mold. Incubate the cell matrix mixture in a standard cell culture incubator at 37 degrees Celsius and 5%carbon dioxide for 60 minutes. When the tissue has formed, carefully soak the mold with four milliliters of ECT culture medium and return the plate to the cell culture incubator for 14 days.Before ECT implantation, you sterilized fine forceps to carefully remove the ECT from the loading posts. Tissue molds can be fabricated with various patterns, post lengths and post spacing to generate different final ECT geometries. For this analysis, a mold with seven millimeter long posts with 2.5 millimeter wide intervals was used.Poloxamer 407 prevents adhesion of the cells to the mold and enables the formation of a characteristic mesh structure, following rapid gel compaction in 14 days. The structure is maintained even after the ECT is released from the mold. The fabricated tissue construct is approximately 1.5 centimeters wide and 0.5 millimeters thick.And the width of each bundle in the mesh is approximately 0.5 millimeters in average. It is possible to generate a 3 centimeter final width mesh ECT containing 24 million cells from a four times larger mold with the same staggered post design. This larger mesh ECT can also be easily removed from the mold and generates a local act of force equivalent to the smaller mesh ECT.It is important to pour the cell matrix mixture evenly into the tissue mold without generating bubbles to prevent feeling defects in the final gel. Functional characterization of the contractor. and fourth frequency relations can be performed using in-built systems.Our ultimate goal is the translation of ECT paradigms into clinical therapies that utilize large animal preclinical studies.

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5.3K Views.  RIKEN Center for Biosystems Dynamics Research. This protocol describes a noble and easy method for creating mesh-shaped engineered cardiac tissues derived from human induced body parts and STEM cell derived cardiac cells. The flexibility of the tissue geometry and the scalability of the preserved tissue function are the main advantages of this technique. The implanted mesh shape ECTs can restore a cardiac structure and function in a rat myocardial infarction model.Confirming its feasibility for use in cardiac regenerative therapy. ...