Name: construction of defined human engineered cardiac tissues to study mechanisms of cardiac cell therapy
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This work was supported by NIH (1F30HL118923-01A1) to T.J.C., NIH/NHLBI PEN contract HHSN268201000045C to K.D.C., the research grant council of Hong Kong TRS T13-706/11(K.D.C), NIH (R01 HL113499) to B.D.G., the American Heart Association (12PRE12060254) to R.J., and Research Grant Council of HKSAR (TBRS, T13-706/11) to R.L. Additional funding was provided to T.J.C. by NIH DRB 5T32GM008553-18 and as a traineeship on NIDCR-Interdisciplinary Training in Systems and Developmental Biology and Birth Defects T32HD075735. The authors also wish to gratefully acknowledge Arthur Autz at The Zahn Center of The City College of New York for assistance with machining the bioreactor and Mamdouh Eldaly for technical assistance. We also thank Dr. Kenneth Boheler for advice on cardiac differentiation, and Dr. Joshua Hare for generously providing human mesenchymal stem cells.

Note: Perform all cell manipulations in aseptic conditions using a HEPA-filtered class II biological safety cabinet and sterilize all solutions by filtering them through a 0.2 &#181;m filter. Perform tissue construction and function testing in either the same aseptic conditions or a laminar flow hood.
1. Seeding of H7 hESCs in Preparation for Cardiac Differentiation
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	<li>(Day 1) Preparing the Basement Membrane Matrix
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		<li>Thaw 150 &#181;l aliquot of hESC qualified basement membra...

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Part A. 17 (23-24), 2973-2980 (2011).</li><li>Lesman, A., 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=Transplantation+of+a+tissue-engineered+human+vascularized+cardiac+muscle.">Transplantation of a tissue-engineered human vascularized cardiac muscle.</a> Tissue Eng. Part A. 16 (1), 115-125 (2010).</li><li>Turnbull, I. C., 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=Advancing+functional+engineered+cardiac+tissues+toward+a+preclinical+model+of+human+myocardium.">Advancing functional engineered cardiac tissues toward a preclinical model of human myocardium.</a> FASEB J. 28 (2), 644-654 (2014).</li><li>Thavandiran, N., Dubois, 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=Design+and+formulation+of+functional+pluripotent+stem+cell-derived+cardiac+microtissues.">Design and formulation of functional pluripotent stem cell-derived cardiac microtissues.</a> Proc. Natl. Acad. Sci. U. S. 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Res. 111 (3), 344-358 (2012).</li><li>Kim, C., 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=Non-cardiomyocytes+influence+the+electrophysiological+maturation+of+human+embryonic+stem+cell-derived+cardiomyocytes+during+differentiation.">Non-cardiomyocytes influence the electrophysiological maturation of human embryonic stem cell-derived cardiomyocytes during differentiation.</a> Stem Cells Dev. 19 (6), 783-795 (2010).</li><li>Dubois, N. C., 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=SIRPA+is+a+specific+cell-surface+marker+for+isolating+cardiomyocytes+derived+from+human+pluripotent+stem+cells.">SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells.</a> Nature Biotechnol. 29 (11), 1011-1018 (2011).</li><li>Hudon-David, F., Bouzeghrane, F., Couture, P., Thibault, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thy-1+expression+by+cardiac+fibroblasts:+lack+of+association+with+myofibroblast+contractile+markers.">Thy-1 expression by cardiac fibroblasts: lack of association with myofibroblast contractile markers.</a> J. Mol. Cell. Cardiol. 42 (5), 991-1000 (2007).</li><li>Gago-Lopez, 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=THY-1+receptor+expression+differentiates+cardiosphere-derived+cells+with+divergent+cardiogenic+differentiation+potential.">THY-1 receptor expression differentiates cardiosphere-derived cells with divergent cardiogenic differentiation potential.</a> Stem Cell Reports. 2 (5), 576-591 (2014).</li><li>Hare, J. M., Traverse, J. 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Cardiol. 54 (24), 2277-2286 (2009).</li><li>Bolli, R., 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=Cardiac+stem+cells+in+patients+with+ischaemic+cardiomyopathy+(SCIPIO):+initial+results+of+a+randomised+phase+1+trial.">Cardiac stem cells in patients with ischaemic cardiomyopathy (SCIPIO): initial results of a randomised phase 1 trial.</a> Lancet. 378 (9806), 1847-1857 (2011).</li><li>Wollert, K. 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Construction of defined human engineered cardiac tissues (hECT) can provide a more consistent and reliable model of human cardiac myocyte function. Critically, all cellular and extracellular components in the system are known and can be manipulated as desired, thus removing the confounding influence of other unknown cell types resulting from the differentiation process. To balance rapid cell growth and high yield, it is preferable that the differentiation starts at 75% confluence of the hESCs, ideally four days after pla...

Cardiac tissue engineering has advanced greatly in the last decade, with multiple groups publishing results of fully functional, beating tissues made from both murine cardiomyocytes1-6 and, more recently, human stem cell-derived cardiac myocytes7-12. The cardiac tissue engineering field is driven by two primary and essentially independent goals: 1) to develop exogenous grafts that can be transplanted into failing hearts to improve function4-6; and 2) to develop in vitro models for studying physiology and disease, or as screening tools for therapeutic development2,7.
Three-dimensional (3-D) cell culture is considered essential for developing next generation screening tools, as the 3-D matrix reflects a more natural cardiac microenvironment than traditional 2-D monolayer cell culture; indeed some aspects of cell biology are fundamentally different in 2-D vs. 3-D cultures13,14. Additionally, engineered cardiac tissues are constructed from completely defined components: an extracellular matrix, and a cell population. For traditional engineered human cardiac tissues, while the extracellular matrix composition (usually fibrin9 or collagen7,8,10) is strictly controlled, the input cell composition is less well defined, with the entire mixture of cells from a directed cardiac differentiation of either embryonic stem cells (ESC7,9) or induced pluripotent stem cells (iPSC10,12) being added to the tissues. Depending on the specific cell line and the efficiency of the differentiation protocol used, the resulting percentage of cardiomyocytes can range from less than 25% to over 90%, the specific cardiomyocyte phenotype (i.e., ventricular-, atrial-, or pacemaker-like) can also vary, even the non-cardiomyocyte fraction can be highly heterogeneous15,16 and alter the maturity of the differentiated cardiac myocytes17.
Recent cardiac tissue engineering work has attempted to control the input population of cells, with either a cardiac reporter human embryonic stem cell line8 or cell surface markers18 being used to isolate the cardiac myocyte component of the differentiation. While initially a tissue composed of only cardiac myocytes would seem to be the ideal, this is in fact not the case; hECTs composed solely of cardiac myocytes fail to compact into functional tissues, with some groups finding a 3:1 ratio of cardiac myocytes:fibroblasts producing the highest twitch force8. By using various cell selection methods, including surface markers for live cell sorting, it is possible to create hECTs with defined cell populations. While markers of non-cardiac stromal cells have been available for some time, such as the putative fibroblast marker CD9019,20, surface markers of cardiac myocytes have been more difficult to identify. SIRP&#945; was among the first cardiac surface markers identified for human cardiac myocytes18 and has been shown to be highly selective for the cardiac lineage. Recently, we have found that double-sorting for SIRP&#945;+ and CD90- cells yields nearly pure cardiomyocytes, with the CD90+ population exhibiting a fibroblast-like phenotype (Josowitz, unpublished observations). Based on these collected findings, herein we describe creating hECTs using a 3:1 combination of SIRP&#945;+/CD90- cardiomyocytes and CD90+ fibroblasts.
The ability to engineer a completely defined human cardiac tissue is essential not only for creating robust screening tools, but also for developing model systems to investigate emerging cell- and gene-based cardiac therapies. In particular, numerous cell therapies for heart failure, utilizing cell types including mesenchymal stem cells (MSC)21, cardiac stem cells22 and bone marrow mononuclear cells23-25, have been tested in clinical trials. While many of the initial results have been promising21,23,25, the initial benefit often diminishes over time26-29. A similar trend has been reported in murine engineered cardiac tissues, which display a significant functional benefit due to MSC supplementation, but the benefit is not sustained during long-term culture1. Underlying the sub-optimal performance is our limited knowledge of the mechanisms governing cell therapies. A deeper understanding of how therapeutic cells exert their beneficial influence, as well as potential negative consequences of myocyte-nonmyocyte interactions, would enable the development of improved therapies yielding clinically significant and sustained benefits, with minimal side effects, for patients with heart failure.
Here, we describe the use of defined hECTs to interrogate mechanisms of cell-based therapy. The controlled tissue composition is essential to identify specific factors impacting cardiomyocyte performance. Directly supplementing hECTs with the therapeutic cell type of interest (e.g., MSCs), can reveal the effects on cardiac myocyte performance, as we have demonstrated in rat ECTs1.
The following multi-step protocol begins with directed cardiac stem cell differentiation, followed by fabrication of the multi-tissue bioreactor, and concluding with a description of tissue construction and functional analysis. Our experiments are performed using the NIH-approved H7 human embryonic stem cell (hESC) line. However, the following protocols have also been tested using an additional hESC line and three induced pluripotent stem cell (hiPSC) lines with similar results. We have found that efficiency in cardiomyocyte differentiation and success in hECT fabrication can be cell line dependent, particularly for hiPSC lines derived from individual patients. By following this protocol, two 6-well dishes are plated with a total of 1.68 million hESCs (140,000 cells per well), which yields approximately 2.5 million myocytes after differentiating for 20 days and sorting, enough to make six defined tissues.

<table border="0">
	<tbody>
		<tr>
			<td>Cell Culture</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma-Aldrich</td>
			<td>A2411</td>
			<td>Prepare a 2.5 mg/ml stock in DMSO and filter-sterilize</td>
		</tr>
		<tr>
			<td>B27 with Insulin</td>
			<td>Life Technologies</td>
			<td>17505055</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 without Insulin</td>
			<td>Life Technologies</td>
			<td>A1895601</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Stemgent</td>
			<td>04-0004</td>
			<td>Create 6 &#956;M stock, then aliquot and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Essential 8 Media</td>
			<td>Life Technologies</td>
			<td>A1517001</td>
			<td></td>
		</tr>
		<tr>
			<td>H7 Human Embryonic Stem Cells</td>
			<td>WiCell</td>
			<td>WA07</td>
			<td></td>
		</tr>
		<tr>
			<td>hESC Qualified Matrix, Corning Matrigel</td>
			<td>Corning</td>
			<td>354277</td>
			<td>Thaw on ice at 4 &#176;C overnight then aliquot 150 &#956;l into separate tubes and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>IWR-1</td>
			<td>Sigma-Aldrich</td>
			<td>I0161</td>
			<td>Create 10 mM stock and aliquot. Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Neonatal Calf Serum</td>
			<td>Life Technologies</td>
			<td>16010159</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-enzymatic Dissociation Reagent: Gentle Cell Dissociation Reagent</td>
			<td>Stem Cell Technologies</td>
			<td>7174</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Life Technologies</td>
			<td>11875-093</td>
			<td>Keep refrigerated</td>
		</tr>
		<tr>
			<td>Y-27632 (ROCK Inhibitor)</td>
			<td>Stemgent</td>
			<td>04-0012</td>
			<td>Resuspend to a 10 mM stock concentration, aliquot and store at -20 &#176;C.&#160; Avoid freeze thaw cycles.</td>
		</tr>
		<tr>
			<td>Cell Sorting</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>4&#8217;,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI)</td>
			<td>Life Technologies</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>CD90-FITC</td>
			<td>BioLegend</td>
			<td>328107</td>
			<td></td>
		</tr>
		<tr>
			<td>Enzymatic Dissociation Reagent: Cell Detach Kit I (0.04 % Trypsin/ 0.03% EDTA, Trypsin neutralization solution and Hanks Buffered Salt Solution)&#160;</td>
			<td>PromoCell</td>
			<td>C-41200</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologics</td>
			<td>S11250</td>
			<td></td>
		</tr>
		<tr>
			<td>SIRP&#945;-PE/Cy7</td>
			<td>BioLegend</td>
			<td>323807</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Construction</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>0.25% Trypsin/0.1% EDTA</td>
			<td>Fisher Scientific</td>
			<td>25-053-CI</td>
			<td>Optional: For collection of supplemental cells of interest</td>
		</tr>
		<tr>
			<td>10x MEM</td>
			<td>Sigma-Aldrich</td>
			<td>M0275-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>10X PBS Packets</td>
			<td>Sigma-Aldrich</td>
			<td>P3813</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen, Bovine Type I</td>
			<td>Life Technologies</td>
			<td>A10644-01</td>
			<td>Keep on ice</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Life Technologies</td>
			<td>11330057</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagles Medium (DMEM), High Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>D5648</td>
			<td></td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3784</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>221465</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>1.5 ml microcentrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>NC0536757</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml polyproylene centrifuge tube</td>
			<td>Corning</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml Polystyrene Round-Bottom Tube</td>
			<td>Corning</td>
			<td>352235</td>
			<td>With integrated 35 &#956;m cell strainer</td>
		</tr>
		<tr>
			<td>50 ml polyproylene centrifuge tube</td>
			<td>Corning</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well flat bottom tissue-culture treated plate</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Scraper, Disposable</td>
			<td>Biologix</td>
			<td>70-2180</td>
			<td></td>
		</tr>
		<tr>
			<td>Polysulfone</td>
			<td>McMaster-Carr</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polytetrafluoroethylene (Teflon)</td>
			<td>McMaster-Carr</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Dissecting Microscope</td>
			<td>Olympus</td>
			<td>SZ-61</td>
			<td>Or similar, must have a mount for the high speed camera to attach</td>
		</tr>
		<tr>
			<td>Electrical Pacing System</td>
			<td>Astro-Med, Inc</td>
			<td>Grass S88X Stimulator</td>
			<td></td>
		</tr>
		<tr>
			<td>High Speed Camera</td>
			<td>Pixelink</td>
			<td>PL-B741U</td>
			<td>Or similar, but must be capable of 100 frames per second for accurate data acquisition</td>
		</tr>
		<tr>
			<td>Plate Temperature Control</td>
			<td></td>
			<td></td>
			<td>Used to maintain media temperature during data acqusition.</td>
		</tr>
		<tr>
			<td>Custom Materials</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>LabView Post-tracking Program</td>
			<td></td>
			<td></td>
			<td>available upon request from the authors</td>
		</tr>
	</tbody>
</table>

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.

To obtain cardiac myocytes, a slightly modified version of the Boheler and Lian differentiation methods is used30,31. It is imperative that the differentiation starts during the log-phase of cell growth, but also that the starting population is sufficiently confluent to obtain a useable number of cells after sorting (approximately 75% is optimal). Typically, for H7 hESCs, plating at a density of 140,000 hESCs per well of a 6-well dish in essential 8 media and 5% CO2 incubator maintained at 37 &#176;...

Watch this Scientific Journal Video about Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy at JoVE.com

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

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 ...

Research

JoVE Journal

Bioengineering

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Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

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Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum

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

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

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Cardiac Spheroids as in vitro Bioengineered Heart Tissues to Study Human Heart Pathophysiology

Despite several advances in cardiac tissue engineering, one of the major challenges to overcome remains the generation of a fully functional vascular ...