Volume 31 Issue 10 - November 15, 2019 PDF
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Human embryonic stem cell–derived cardiomyocytes restore function in infarcted hearts of non-human primates
Yen-Wen Liu1,2,3,*, Billy Chen1,2,4, Xiulan Yang1,2,5, James A Fugate1,2,5, Faith A Kalucki1,2,5, Akiko Futakuchi-Tsuchida1,2,5, Larry Couture6, Keith W Vogel7, Clifford A Astley7, Audrey Baldessari7, Jason Ogle7, Creighton W Don4, Zachary L Steinberg4, Stephen P Seslar4,8, Stephanie A Tuck1,2,5, Hiroshi Tsuchida1,2,5, Anna V Naumova1,2,9,10, Sarah K Dupras1,2,5, Milly S Lyu9, James Lee4 , Dale W Hailey1, Hans Reinecke1,2,5, Lil Pabon1,2,5, Benjamin H Fryer1,2,5, W Robb MacLellan1,2,4, R Scott Thies1,2,5 & Charles E Murry1,2,4,5,11
1 Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, Washington, USA.
2 Center for Cardiovascular Biology, University of Washington, Seattle, Washington, USA.
3 Division of Cardiology, Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
4 Department of Medicine/Cardiology, University of Washington, Seattle, Washington, USA.
5 Department of Pathology, University of Washington, Seattle, Washington, USA.
6 City of Hope, Beckman Research Institute, Duarte, California, USA.
7 Washington National Primate Research Center, University of Washington, Seattle, Washington, USA.
8 Department of Pediatrics, University of Washington, Seattle Children’s Hospital, Seattle, Washington, USA.
9 Department of Radiology, University of Washington, Seattle, Washington, USA.
10 Research Institute of Biology and Biophysics, National Research Tomsk State University, Tomsk, Russia.
11 Department of Bioengineering, University of Washington, Seattle, Washington, USA.
 
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Here our research team, including Associate Professor Dr. Yen-Wen Liu at National Cheng Kung University (NCKU) Hospital, College of Medicine, NCKU in Tainan, Taiwan and Researchers at University of Washington (UW) Medicine in Seattle, Washington, U.S. present exciting data on cardiac cell therapy. For the first time in a non-human primate (Macaca nemestrina) model, we have successfully used human embryonic stem cell (ESC)-derived cardiomyocytes (hESC-CMs) to repair cardiac function in monkeys with heart failure cause by myocardial infarction (MI).1 The hESC-CMs form new cardiac muscle that integrates into macaque heart so that the failing monkey heart pumps vigorously again. In some animals, the cells returned the hearts’ contractile functioning to almost normal levels. Moreover, we only observed only one of five hESC-CM-transplanted monkeys experiencing graft-associated ventricular arrhythmias. We did not find any tumor formation in macaques after cell therapy. In short, our findings showed that hESC-CMs can re-muscularize infarcts in monkey hearts and, in doing so, reduce scar size and restore a significant amount of heart function. These findings suggest that hESC-CMs transplanted into the post-infarct failing hearts will result in long-term therapeutic benefit with an acceptable risk profile. Thus, owing to the high translational value of the macaque model, our data demonstrated the important clinical implications.

Most heart failure is caused by the death of heart muscle due to heart attacks. Because heart muscle does not regenerate, the damaged areas will be replaced with scar tissue, which does not contract. As a result, the heart becomes weaker and can no longer pump enough blood to supply other organs with the oxygen and nutrition it needs to function. This is called heart failure (HF). HF symptoms include fatigue, profound weakness and shortness of breath. HF is a global and major health threat.2 More than 25 million people worldwide live with heart failure, and more than 600,000 die of this disease each year in U.S. Currently, neither pharmacological nor interventional therapy could restore the heart’s lost muscle function. Therefore, regenerative medicine should be able to offer opportunities to remuscularize the failing hearts.

In this study1, we induced experimental heart attacks in the pig-tailed monkeys. Most notably, the non-human primate model used in this study is highly informative. Although the macaque heart is relatively small and even marginally larger than large rabbit, because of macaque’s evolutionary proximity to humans, its cardiovascular anatomy and physiology, such as heart rate, blood pressure, susceptibility to arrhythmia etc., is similar to that of the human heart. The heart attacks reduced the hearts’ left ventricular ejection fraction — the amount of blood being pumping out from the heart per beat— from about 65 percent to 40 percent, enough to put these animals into heart failure (Figure 1a). In contrast to our previous study3, this time the induced heart-attack injury was severe enough to compromise the heart. Two weeks after infarction, we intramuscularly injected 750 million hESC-CMs into the infarct and peri-infarct region. For comparison, a control group was injected with a cell-free version of the solution that was used to inject the stem cells into the treatment animals. Four weeks after treatment, ejection fraction of the untreated control animals remained unchanged: it stayed at about 40%. However, in the treated animals, the ejection fraction had risen to ~50%, about half-way back to normal (Figure 1a). Cardiac magnetic resonance imaging scans showed that new heart muscle had grown within what had been scar tissue in the treated hearts, while no new muscle was seen in the untreated animals (Figure 1b). Two hESC-CM-treated animals and one control animal were monitored for three months. The ejection fraction in the control animal declined, whereas the treated animals continued to improve: Their ejection fractions rose from 51% at four weeks after treatment to 64% at three months (Figure 1c). The transplanted hESC-CMs had formed new cardiac muscle tissue in the infarcted region. The new cardiac muscle replaced 10~29% of the scar tissue, integrated with the surrounding healthy tissue and developed into mature heart cells (Figure 1d and 1e). It is worth mention that the functional recovery of the infarcted heart seen in this study was larger than previous studies using rat4 or guinea pig5 models of myocardial infarction. This could be due to the greater physiological match between human and macaque. Thus, the therapeutic benefit may be further increased when human cardiomyocytes are transplanted into the failing human hearts.
Figure 1. Therapeutic benefit of hESC-CM transplantation on post-infarct cardiac function. a. LVEF is comparable between groups before myocardial infarction and at post-infarct baseline (p >0.05) but shows a significant improvement after hESC-CM treatment (p = 0.004). b. Representative cardiac MRI at end-diastolic and end-systolic phases of the cardiac cycle at 4 weeks after hESC-CM treatment. At baseline, the infarct region is a homogeneous tissue located in the anterior wall and interventricular septum. At 1 month and 3 months after cells engraftment, new areas of visible dark (post-Gd infarct panel) tissue appear within the infarct (arrows). c. Extending survival to 12 weeks demonstrates a modest reduction in LVEF in control hearts and further improvement with hESC-CM treatment. This indicates the benefits seen at 4 weeks are stable with the potential for substantial further improvement. d & e. Low magnification immunofluorescent images stained for cardiac troponin T (cTnT; red; human + monkey myocardium), human-specific cardiac troponin I (human cTnI; green; human myocardium) and type I collagen to identify scar tissue (blue). d. Control heart showing transmural infarct and lateral border zone. Note the viable rim of host subendocardial myocardium and the thinned infarct wall compared to the border zone. Scale bar, 5 mm. f. hESC-CM engrafted heart showing large islands of human myocardium (green) within the infarct and lateral border zone. Scale bar, 5 mm.

Compared to previous studies of transplanted ESC-CMs in non-human primate using the small infarct model,3, 6 there were two notable difference. First, both the cell-treated animals and the vehicle-treated animals (as the controls) had significantly more post-infarct ventricular arrhythmias (Figure 2a). This could be due either to the larger myocardial infarction or the omission of the amiodarone from this study. Second, there was no statistical difference in the number or duration of ventricular arrhythmias between these two groups either before or after intramuscular injection. To further investigate the ventricular arrhythmias, we did catheter-based electrophysiological studies. A standard, programmed, ventricular-stimulation protocol was used to induce ventricular tachycardia during the electrophysiological studies. We found that one hESC-CM-treated monkey experienced graft-associated ventricular tachycardia, shown by electrophysiological study to originate from a point-source acting as an ectopic pacemaker. In the catheter-based electrophysiological study, ventricular arrhythmia inducibility and severity were graded as previously reported.7 Neither the arrhythmia inducibility nor the severity significantly differed between the hESC-CM-treated and control monkeys (Figure 2b). Therefore, intramyocardial injection of hESC-CMs did not increase the risk and the severity of inducible ventricular arrhythmias.
Figure 2. Analysis of ventricular arrhythmias. a. Spontaneous ventricular arrhythmias in large infarct protocol (3-h mid-LAD occlusion) for four control animals (blue) and five hESC-CM-treated animals (red) were recorded as h/d (24 h). Both groups have ventricular arrhythmias before and after injection (day 0), but the hESC-CM group had one macaque with protracted ventricular arrhythmias (arrow), that likely are treatment-related. b. Programmed electrical stimulation studies demonstrate that the inducibility and severity between control (N = 3) and hESC-CM-treated hearts (N = 5) were not significantly different (p = 0.816, two-tailed t-test, df = 6 for inducibility; p = 0.411, two-tailed t-test, df = 6 for severity).

Our data indicate that hESC-CMs transplanted into the post-infarct failing heart may mediate long-term therapeutic benefit with an acceptable risk profile.

References
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  7. Chelsky LB, Cutler JE, Griffith K, Kron J, McClelland JH, McAnulty JH. Caffeine and ventricular arrhythmias. An electrophysiological approach. JAMA. 1990;264:2236-2240.
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