Name: implantation of hipsc-derived cardiac-muscle patches after myocardial injury in a guinea pig model
Uploaded: 2019-03-18T13:00:00
Description: Watch this Scientific Journal Video about Implantation of hiPSC-derived Cardiac-muscle Patches after Myocardial Injury in a Guinea Pig Model at JoVE.com

No funding was received for this study

Animals received humane care in compliance with the Guide for the Principles of Laboratory Animals, prepared by the Institute of Laboratory Animal Resources, and published by the National Institutes of Health. All animal protocols were approved by the responsible local authority (&#8216;&#8216;Amt f&#252;r Gesundheit und Verbraucherschutz, Hansestadt Hamburg&#8217;&#8217;/ Animal protocol # 109/16).
1. Obtain Animals
<ol>
	<li>Commercially obtain female Guinea pigs weighing 500&#8211;600 g.<...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+applications+for+human+pluripotent+stem+cells.">Cardiac applications for human pluripotent stem cells.</a> Current pharmaceutical design. 15, 2791-2806 (2009).</li><li>Shiba, Y., 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+ES-cell-derived+cardiomyocytes+electrically+couple+and+suppress+arrhythmias+in+injured+hearts.">Human ES-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts.</a> Nature. 489, 322-325 (2012).</li><li>Chong, 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=Human+embryonic-stem-cell-derived+cardiomyocytes+regenerate+non-human+primate+hearts.">Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts.</a> Nature. 510, 273-277 (2014).</li><li>Kattman, S. 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=Stage-specific+optimization+of+activin/nodal+and+BMP+signaling+promotes+cardiac+differentiation+of+mouse+and+human+pluripotent+stem+cell+lines.">Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines.</a> Cell stem cell. 8, 228-240 (2011).</li><li>Watanabe, T., Rautaharju, P. M., McDonald, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventricular+action+potentials,+ventricular+extracellular+potentials,+and+the+ECG+of+guinea+pig.">Ventricular action potentials, ventricular extracellular potentials, and the ECG of guinea pig.</a> Circulation research. 57, 362-373 (1985).</li><li>Weinberger, F., 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+repair+in+guinea+pigs+with+human+engineered+heart+tissue+from+induced+pluripotent+stem+cells.">Cardiac repair in guinea pigs with human engineered heart tissue from induced pluripotent stem cells.</a> Science translational medicine. 8, 363ra148 (2016).</li><li>Shiba, Y., 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=Electrical+Integration+of+Human+Embryonic+Stem+Cell-Derived+Cardiomyocytes+in+a+Guinea+Pig+Chronic+Infarct+Model.">Electrical Integration of Human Embryonic Stem Cell-Derived Cardiomyocytes in a Guinea Pig Chronic Infarct Model.</a> Journal of Cardiovascular Pharmacology and Therapy. 19, 368-381 (2014).</li><li>van Laake, L. W., 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+cardiomyocytes+survive+and+mature+in+the+mouse+heart+and+transiently+improve+function+after+myocardial+infarction.">Human embryonic stem cell-derived cardiomyocytes survive and mature in the mouse heart and transiently improve function after myocardial infarction.</a> Stem cell research. 1, 9-24 (2007).</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>Johns, T. N., Olson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+myocardial+infarction.+I.+A+method+of+coronary+occlusion+in+small+animals.">Experimental myocardial infarction. I. A method of coronary occlusion in small animals.</a> Annals of surgery. 140, 675-682 (1954).</li><li>van den Bos, E. J., Mees, B. M., de Waard, M. C., de Crom, R., Duncker, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+model+of+cryoinjury-induced+myocardial+infarction+in+the+mouse:+a+comparison+with+coronary+artery+ligation.">A novel model of cryoinjury-induced myocardial infarction in the mouse: a comparison with coronary artery ligation.</a> Heart and circulatory physiology. , H1291-H1300 (2005).</li></ol>

A variety of small animal models are available to study the effect that cell transplantation exerts on injured hearts9,10,11. We chose a guinea pig model because of all small animal models its (electro)physiology resembles most closely that of humans. The advantages of small animal models are simple housing, manageable costs, and few workforces. In comparison with mice and rats, guinea pigs&#180; cardiac (electro)physiology is m...

The number of patients with heart failure is increasing in our aging population. For end-stage heart failure, orthotopic heart transplantation is the only curative treatment option. However, especially in European countries, there is an increasing organ donor shortage. Therefore, alternative treatment options are necessary. Recent achievements in mechanical circulatory support are promising, but especially in the long-term run, limited by its side effects like bleeding, pump thrombosis and infectious complications1.
The endogenous regeneration capacity of the adult human heart is extremely limited. Therefore, cardiac regeneration therapies might become an alternative treatment option for end-stage heart failure patients2,3. Different techniques including stem cell-based cell injection or tissue engineering approaches have been described3,4,5.
Human induced pluripotent stem cells (hiPSC), as well as human embryonic stem cells (hESC) can be effectively differentiated to spontaneously beating human cardiomyocytes6, which has been a major achievement in the field of cardiac regenerative therapies.
To replace myocardium after a myocardial infarction and to improve the function of a failing heart, survival of a suitable number of cardiomyocytes and their mechanical and electrical coupling with the native heart is essential. To investigate the potential of cardiac regenerative therapies with human iPS cell derived cardiomyocytes, a suitable research model is needed. The ideal model should be cost-effective and have a human-like physiology and electrophysiology. Large animal models like pigs would be ideal from that point of view, however, those experiments are very expensive and large amounts of cardiomyocytes would be necessary to replace a relevant number of cardiomyocytes in order to see effects on the left ventricular function in a pig infarction model.
To answer elementary biological questions towards human cell-based cardiac regeneration, e.g., cell survival, vascularization, and electrical coupling, small animal models are more suitable. From the available small animal models, the guinea pig is the most useful species, as compared to rats and mice, as their electrophysiology more closely resembles the situation in humans7. In this guinea pig model, we induced a transmural cryoinjury of the left ventricle. One week after induction of myocardial infarction implantation of a three-dimensional, spontaneously beating hiPS-cell derived cardiomyocyte patch was performed. Cardiomyocyte cell survival was evaluated 28 days after implantation by histological examination.

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implantation of hipsc-derived cardiac-muscle patches after myocardial injury in a guinea pig model

Due to the limited regeneration capacity of the heart in adult mammals, myocardial infarction results in an irreversible loss of cardiomyocytes. This loss of relevant amounts of heart muscle mass can lead to the heart failure. Besides heart transplantation, there is no curative treatment option for the end-stage heart failure. In times of organ donor shortage, organ independent treatment modalities are needed. Left-ventricular assist devices are a promising therapy option, however, especially as destination therapy, limited by its side-effects like stroke, infections and bleedings. In recent years, several cardiac repair strategies including stem cell injection, cardiac progenitors or myocardial tissue engineering have been investigated. Recent improvements in cell biology allow for the differentiation of large amounts of cardiomyocytes derived from human induced pluripotent stem cells (iPSC). One of the cardiac repair strategies currently under evaluation is to transplant artificial heart tissue. Engineered heart tissue (EHT) is a three-dimensional in vitro created cardiomyocyte network, with functional properties of native heart tissue. We have created EHT-patches from hiPSC derived cardiomyocytes. Here we present a protocol for the induction of left ventricular myocardial cryoinjury in a guinea pig, followed by implantation of hiPSC derived EHT on the left ventricular wall.

This guinea pig model is a suitable model to investigate cardiac regeneration after implantation of hiPSC derived EHT-patches. It reproducibly leads to large transmural myocardial injuries. Scar size is evaluated by histology four weeks after cryoinjury. Mason trichrome staining reveals large transmural scars (Figure 2). Scar size was similar over a large number of injured animals reflecting a high degree of reproducibility8. On average 25% of the left ventricular myo...

Watch this Scientific Journal Video about Implantation of hiPSC-derived Cardiac-muscle Patches after Myocardial Injury in a Guinea Pig Model at JoVE.com

Implantation of hiPSC-derived Cardiac-muscle Patches after Myocardial Injury in a Guinea Pig Model

Here we present a protocol for the induction of left ventricular cryoinjury followed by the implantation of a cardiac muscle patch, derived from human iPS-cell cardiomyocytes in a guinea pig model.

Due to the limited regeneration capacity of the heart in adult mammals, myocardial infarction results in an irreversible loss of cardiomyocytes. This loss ...

The aim of the method is to evaluate the functional consequences of engineered heart tissue implantation in a small animal cryoinfarction model. The advantage of this method is that it allows for the induction of relatively reproducible myocardial infarctions with stable infarction sizes. After confirming a lack of response to toe pinch, place the anesthetized guinea pig on a 40 to 42 degrees Celsius warming platform in the supine position.Apply eye ointment and shave and depilate the thorax. Apply 25 degrees Celsius ultrasound transducer gel to the exposed skin and place the transducer from an echocardiography system onto the thorax to acquire two-dimensional parasternal long axis views in B mode from the right neck toward the left leg at the plane of the aortic valve with a concurrent visualization of the left ventricle apex to investigate the pre-operative left ventricular function. Then turn the transducer 90 degrees to obtain a short axis B mode view at the mid papillary level.After the last image has been obtained, transfer the guinea pig to the surgical table and tape the limbs in a spread eagle position. Disinfect the exposed skin with two sequential iodine-based and 80%ethanol scrubs and make a 1.5 centimeter vertical incision in the tracheal area. Bluntly dissect the muscles covering the trachea.When the trachea can be observed, puncture the air tube with an 18 gauge IV cannula and insert the flexible part of the cannula as a tracheal tube. Connect the tracheal tube to an animal respirator for continuous ventilation during the procedure and count the rib spaces starting at the first intercostal space to locate the fifth intercostal space. Make a two centimeter horizontal incision on the fifth intercostal space on the left side of the guinea pig and remove the subcutaneous tissues before dissecting the muscles with an electrocautery until the intercostal muscles are reached.Gently dissect the intercostal muscles with tweezers and small scissors until the pleural space is reached and the left lung can be visualized. Insert a retractor between the ribs and open the retractor carefully until a good view of the heart is obtained. Open the pericardium approximately one centimeter in the region of the anterior left ventricular wall and place a compress on the left lung to protect the lung from damage when inducing the cryoinjury of the left ventricle.Next, cool the tip of an eroded metal stamp with a cross-sectional diameter of 0.5 centimeters in liquid nitrogen for three minutes before pressing the probe onto the left anterior wall of the heart for 30 seconds. Then place a 200 degrees Celsius electric soldering iron inside the stamp to warm the stamp so that the metal can be removed from the tissue. After the third stamp application, remove the retractor from the intercostal space and clamp the outflow tube of the ventilator for two seconds to inflate the lungs with maximum pressure to avoid atelectasis of the lung.Then close the ribs with two 4-0 sutures, the muscles over the ribs with a 5-0 running suture and the skin with 5-0 suture single stitches. After removing the tracheal tube, use a single 8-0 suture to close the puncture site at the trachea and close the wound with three single stitch 5-0 sutures. Seven days after cryoinjury, remove sutures and make a two centimeter horizontal skin incision in the scar area of the left lateral side and use electrocautery to gently dissect the extra pleural adhesions.Carefully open the pleural space with scissors and insert a rib spreader to expose the heart. Visually identify the region of the infarction by its pale color compared to the healthy surrounding myocardium and place an engineered heart tissue patch over the infarction region. Secure the patch with two 8-0 sutures at both sides in the non-infarcted area of the heart and inflate the lungs with pressure to avoid atelectasis of the lungs.Then remove the retractor from the intercostal space and close the animal as demonstrated. Four weeks after engineered heart tissue implantation, perform transthoracic echocardiography as demonstrated to evaluate changes in the left ventricle function overtime. Mason trichrome staining four weeks after cryoinjury reveals large transmural scars over 25%of the left ventricular myocardium on average.Dystrophin staining demonstrates large myocardial grafts that have partially re-muscularized to the scar and staining for human Ku80 that confirms the human origin of the newly formed myocardium. Transthoracic echocardiography monitoring of the implanted engineered heart tissue reveals an improvement in the left ventricle ejection fraction, fractional area shortening, and a decrease in left ventricular and diastolic diameter. It is essential to carefully open the pleural cavity in this kind of re-do operation as adhesions between the thoracic wall and the left ventricle can be present and there's a certain risk to injure the left ventricular myocardium.After the implantation of the human engineered heart tissue, several methods can be used to monitor for changes in left ventricular ejection fraction. Besides echocardiography, an invasive measurement of pressure volume loops can be used to monitor those changes. This method allows to investigate all aspects of human engineered tissue implantation.Functional consequences as well as potential risks like arrhythmias can be evaluated in this small animal model.

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