Published online before print April 24, 2008, doi: 10.1161/CIRCRESAHA.108.175505
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© 2008 American Heart Association, Inc.
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Human Embryonic Stem Cell–Derived Cardiomyocytes and Cardiac Repair in Rodents
From the Hubrecht Institute (L.W.v.L., R.P., C.L.M.), Utrecht; Heart Lung Center Utrecht (L.W.v.L., P.A.D.); and Department of Anatomy and Embryology (R.P., C.L.M.), Leiden University Medical Center, The Netherlands.
Correspondence to C. L. Mummery, Hubrecht Institute (Developmental Biology and Stem Cell Research), Uppsalalaan 8, 3584 CT Utrecht, The Netherlands. E-mail c.mummery{at}niob.knaw.nl
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Cell transplantation may restore heart function in disease associated with loss or dysfunction of cardiomyocytes. Recently, Laflamme et al reported an improvement in cardiac function in immunodeficient rats 4 weeks after coronary artery ligation and injection of human embryonic stem cell–derived cardiomyocytes (hESC-CMs). We have recently carried out a comparable study transplanting hESC-CMs to the hearts of mice with myocardial infarction. Our findings were similar up to the 4-week time point, with significant improvements in cardiac function. However, our follow-up was longer, and, at 3 months, the difference between mice receiving cardiomyocytes and those receiving other cells was no longer significant. Hypothesizing that the improvement observed by Laflamme et al may have been more likely to be sustained long term because the grafts in their study appeared larger, we injected 3 times as many cells. Although this resulted in a significantly increased graft size, we again observed a functional improvement at 1 month but not at 3 months. Our results show that midterm data in these kinds of experiments must be interpreted with caution and longer-term follow-up is essential to draw conclusions on the efficacy of cardiac cell transplantation. Furthermore, our findings demonstrate the unlikely success of merely generating and injecting more cells of the same type to increase functional improvement.
Key Words: cell transplantation • MRI • myocardial infarction • stem cells
Human embryonic stem cell–derived cardiomyocytes (hESC-CMs) may have potential in replacing cardiomyocytes that are lost or dysfunctional in cardiac disease, such as myocardial infarction. Recently, Laflamme et al reported an improvement in cardiac function in immunodeficient rats 4 weeks after coronary artery ligation and injection of hESC-CMs 4 days later.1 In their careful evaluation, cardiomyocyte-specific effects were distinguished from general cell-based benefit by including a control group of rats that had received noncardiomyocyte derivatives from the same hESC line. This comparison showed that the observed functional enhancement was specifically attributable to cardiomyocyte-enriched differentiated hESCs.
Nevertheless, we would suggest a note of caution based on our own longer-term analysis of cardiac function in mice that have undergone similar transplantation with hESC-CMs. In that recently published study,2 we injected cardiomyocyte-enriched differentiated hESCs into uninjured and acutely infarcted hearts of immunodeficient NOD-SCID mice and performed a longitudinal follow-up of cardiac function by MRI.3 Like Laflamme et al, we found that hESC-CMs survived selectively and improved heart function 4 weeks after myocardial infarction when compared to noncardiac differentiated derivatives of hESCs.2 However, at the 12-week time point, cardiac function was not improved compared to the control animals,2 even though only cardiomyocytes had formed a substantial graft in the infarcted heart (Figure 1): 80% to 95% of the surviving donor cells had a cardiomyocyte phenotype. The remaining cells expressed the endoderm marker cytokeratin 8 (recognized by Troma-1). Only rarely (<1%) were cells positive for the endothelial cell marker von Willebrand factor (Figure I in the online data supplement, available at http://circres.ahajournals.org) or smooth muscle actin, whereas staining of the grafts for stem cell markers (Tra-1-60, Oct-4, and Sox-2) was negative in all cases.
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-actinin; blue, DNA (DAPI). Scale bars: 100 µm (a and b); 10 µm (c and d).We quantified graft size based on a semiquantitative scaling (scale from 0 to 13) based on green fluorescent protein (GFP) fluorescence in the whole heart by a blinded investigator. In a subset of hearts (n=14), the absolute number of GFP fluorescent cells was also counted in sections by a different blinded investigator. We, thus, found that a semiquantitative score of 1 to 3 corresponded to 100 to 1000 cells, 4 to 6 to 1000 to 10 000 cells, and >7 to 10 000 to 100 000 cells.
A reduction in graft size from the 4- to the 12-week time point could account for the transient improvement in cardiac function. However, in a separate group of mice that underwent the same procedures but were euthanized after 4 weeks, the graft size was not significantly larger than that after 12 weeks (mean semiquantitative score, 5.75±0.92 versus 3.75±0.72, P=0.14, n=8 to 12 per group). We, therefore, repeated our initial study but now injecting 3 million cardiomyocyte-enriched differentiated hESCs intramyocardially at 3 different sites in the infarct and border zone, instead of 1 million cells in 1 injection. As expected, the total graft size increased proportionally (Figure 2). Although the resulting initial preservation of cardiac function, as measured by MRI, was present at 4 weeks, after a 12 weeks of follow-up, this had become insignificant in comparison with the control group (Figure 3a). To exclude the possibility that this was attributable to a negative effect of the injections themselves, we also injected infarcted mice with vehicle, or completely left out the injection step, and found no negative effect of intramyocardial injection on the function of the infarcted heart (supplement Table I). In addition, injection of either cardiomyocyte enriched– or cardiomyocyte negative– differentiated hESCs in infarcted mice improved their long-term survival compared to uninjected controls (Figure 3b).
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Some differences between ours and the study by Laflamme et al remain, as discussed by Rubart and Field,4 including the timing of cell injection and the use of a prosurvival cocktail. In the study by Laflamme et al, but not ours, the addition of prosurvival factors was necessary for graft survival in the infarcted heart. Although we demonstrate that a potential difference in graft size is unlikely to affect long-term outcome in these types of studies, there is a possibility that 1 or more components of the acute survival cocktail used would improve long-term myocyte function. Moreover, stimulating blood supply to the grafts may be necessary to improve donor cell function.
Our results show that midterm data in these kinds of experiments must be interpreted with caution and that longer-term follow-up is essential to draw conclusions on the efficacy of cardiac cell transplantation. Furthermore, our findings demonstrate that the expectation of sustained functional improvement by merely generating and injecting more cells of the same type is unlikely.
Nevertheless, both studies warrant optimism because of manifest graft survival, and with refined application methods or engineered scaffolds, stem cell–derived cardiomyocytes could be expected to restore long-term contractility to the damaged heart.
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This work is supported by the European Community’s Sixth Framework Programme contract (‘HeartRepair’) LSHM-CT-2005-018630 (to L.W.v.L, R.P.) and by the Interuniversity Cardiology Institute of the Netherlands postdoctoral fellowship 2007-2008 (to L.W.v.L.).
Disclosures
None.
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Original received March 12, 2008; revision received April 1, 2008; accepted April 10, 2008.
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1. Laflamme MA, Chen KY, Naumova AV, Muskheli V, Fugate JA, Dupras SK, Reinecke H, Xu C, Hassanipour M, Police S, O’sullivan C, Collins L, Chen Y, Minami E, Gill EA, Ueno S, Yuan C, Gold J, Murry CE. Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nat Biotechnol. 2007; 25: 1015–1024.[CrossRef][Medline] [Order article via Infotrieve]
2. van Laake LW, Passier R, Monshouwer-Kloots J, Verkleij AJ, Lips DJ, Freund C, Den Ouden K, Ward-van Oostwaard D, Korving J, Tertoolen LG, van Echteld CJ, Doevendans PA, Mummery CL. Human embryonic stem cell-derived cardiomyocytes survive and mature in the mouse heart and transiently improve function after myocardial infarction. Stem Cell Res. 2007; 1: 9–24.
3. van Laake LW, Passier R, Monshouwer-Kloots J, Nederhoff MG, Ward-van Oostwaard D, Field LJ, van Echteld CJ, Doevendans PA, Mummery CL. Monitoring of cell therapy and assessment of cardiac function using magnetic resonance imaging in a mouse model of myocardial infarction. Nat Protoc. 2007; 2: 2551–2567.[CrossRef][Medline] [Order article via Infotrieve]
4. Rubart M, Field LJ. ES cells for troubled hearts. Nat Biotechnol. 2007; 25: 993–994.[CrossRef][Medline] [Order article via Infotrieve]
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