Large Stem Cell–Derived Cardiomyocyte Grafts: Cellular Ventricular Assist Devices?
Authors: Sian E Harding
Journal: Molecular Therapy (2014)

While the battle rages on about the endogenous repair capacity in the heart, solid work is progressing to take the promising exogenous pluripotent stem cells toward a clinical application. We know that the human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) can reliably be differentiated into cardiac myocytes; protocols are being defined, refined, and scaled up continuously. However, the hard grind and expense of producing the bulk of cardiomyocytes required, delivering them in a way that enables retention and using a clinically relevant model, is demanding and, arguably, unglamorous work. Certainly, Geron appears to have held that opinion when it abandoned the effort as unprofitable in 2011. In their recent Nature paper,1Chong JJ Yang X Don CW Minami E Liu YW Weyers JJ et al.Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts.Nature. 2014; (e-pub ahead of print 30 April 2014)Google Scholar Murry and Laflamme's group report important progress toward this goal by producing significant quantities of muscle in the hearts of macaque monkeys using hESC-derived cardiomyocytes (hESC-CMs). Although this is not a definitive study in that the small numbers of animals did not allow statistical analysis of functional outcomes, the authors made several key observations. First, it was logistically possible to introduce the hESC-CMs via the relatively straightforward method of administering multiple intramyocardial injections and to produce a functionally relevant amount of tissue. The issue of cell loss was addressed simply by massively increasing the number of hESC-CMs used (to more than 1 billion cells), and the authors were able to scale up their processes sufficiently to do this with the aid of cryopreservation protocols. They demonstrated coupling to the host heart in terms of both cardiomyocyte excitation–contraction coupling and infiltration of the host vasculature to support the graft. Less encouragingly, they detected arrhythmias in the presence of the graft; however, this is not an unexpected event, and the nature and trajectory of the arrhythmias observed give important information that can lead to a more measured response than simply rejecting the entire hESC-CM strategy. A central feature of the study was the choice of the monkey as a model; excitation–contraction coupling varies widely between species, and the use of rodents (with heart rates 5- to 10-fold higher than humans) to assess integration of human stem cell–derived cardiomyocytes has always been a concern. Reports of grafts being kept isolated from surrounding mouse myocardium by encapsulating layers of fibrous tissue may represent the death of those human cardiomyocytes that did couple to the host.2van Laake LW Passier R Doevendans PA Mummery CL Human embryonic stem cell–derived cardiomyocytes and cardiac repair in rodents.Circ Res. 2008; 102: 1008-1010Crossref PubMed Scopus (196) Google Scholar One of the main aims of grafting experiments is to detect potential arrhythmic events so as to study their underlying mechanisms. Arrhythmias may arise from spontaneous pacemaker activity from the graft, poor coupling of host/graft cells, or mismatched properties due to immaturity of hESC-CMs or species differences. In this study, the elegant use of an hESC line engineered to express a calcium reporter clearly showed the coupling of graft hESC-CMs to the host heart. To pinpoint the contribution from the known immaturity of the hESC-CMs, it is necessary to reduce the differences attributable to species incompatibility. Nonhuman primates are the most appropriate choice for this—and were vital before the transition to humans. Murry and Laflamme's team previously used an intermediate model, the guinea pig, which has an action-potential morphology and calcium-cycling properties more similar than those of the rat/mouse to human heart.3Shiba Y Fernandes S Zhu WZ Filice D Muskheli V Kim J et al.Human ES-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts.Nature. 2012; 489: 322-325Crossref PubMed Scopus (549) Google Scholar Notably, this model did not display the same postgrafting arrhythmic tendencies that the primate model did. This difference was ascribed to the greater possibility for arrhythmia-generating re-entrant circuits in larger hearts. Another difference is that, for the guinea pig, the model of disease used was cryoinjury, which produces a discrete area of tissue death. The guinea pig has a high degree of collateral circulation, so tying off a vessel to mimic the occluding event in human does not produce an infarct. This model in the primate was produced by transient occlusion and so was closer to the clinical situation, producing a scar that (as in humans) has residual transmural islands of cardiomyocyte tissue. This underlines the importance of matching not only the species but the pathology to produce a true model of clinical events. The choice of embryonic over induced pluripotent stem cells (hiPSCs) might be considered scientifically less than ideal as well as unnecessarily contentious from an ethical standpoint, especially when combined with nonhuman primate studies. However, this group's long experience with the hESC lines in question, the assurance of genetic stability, and the work that went into differentiation protocols are powerful arguments supporting these cell lines for first proof-of-concept studies. Once differentiated, hESC-CMs and hiPSC-CMs share an immature phenotype, so the use of hESC-CMs is likely to be a good predictor for both cell types. Advantages of autologous use of hiPSCs in terms of overcoming immune rejection remain theoretical (and contested), and there is anecdotal experience that differentiation protocols have more variable outcomes on hiPSC lines. Looking to the future, it would seem more likely that the hiPSC would be the final product for clinical use, but in fact clinical trials for spinal cord injury and retinal degeneration are already proceeding with hESCs.4Bretzner F Gilbert F Baylis F Brownstone RM Target populations for first-in-human embryonic stem cell research in spinal cord injury.Cell Stem Cell. 2011; 8: 468-475Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar,5Schwartz SD Hubschman JP Heilwell G Franco-Cardenas V Pan CK Ostrick RM et al.Embryonic stem cell trials for macular degeneration: a preliminary report.Lancet. 2012; 379: 713-720Abstract Full Text Full Text PDF PubMed Scopus (1102) Google Scholar It is further notable for this study that, although the hESC-CMs were pretreated briefly with a prosurvival cocktail, no attempt was made to improve their maturity. This may reflect the sheer difficulty of applying to these enormous quantities of cells the various proposed maturation strategies, such as aging, transfection, or development of three-dimensional constructs. Alternatively, it might be taking advantage of the immature phenotype that confers a resistance to the initially hypoxic conditions of the graft before vascularization has occurred. An interesting side note is the authors’ mention that tissue-engineered patches were used in different areas at the same time that the dissociated hESC-CMs were injected but were less successful. The possibility of comparing multiple strategies within an experiment is a powerful advantage of the use of large hearts. Is the occurrence of arrhythmia an insuperable barrier to progression toward the clinic? The evidence collected by Murry and Laflamme's team suggests that, even with the best species match and the appropriate disease model, intrinsic properties of the graft will produce arrhythmic events. Indeed, it is difficult to imagine that introducing such a large amount of active beating tissue to the host heart would not have consequences. It is important to note, however, that none of these events was fatal. Unlike the previous experience with skeletal myoblasts,6Murry CE Field LJ Menasche P Cell-based cardiac repair: reflections at the 10-year point.Circulation. 2005; 112: 3174-3183Crossref PubMed Scopus (314) Google Scholar there is at least the potential for hESC-CMs or hiPSC-CMs to mature toward an adult cardiomyocyte phenotype in the in vivo environment. There are indications in this study that maturation had begun to occur, and arrhythmias declined toward the end of the 3-month study period. One key goal arising from the study for the scientific community could be the development of strategies to mitigate arrhythmic events during the initial period of vulnerability while the hESC-CMs or hiPSC-CMs mature in vivo. From the clinical side, protection of a patient from arrhythmia or other adverse events is increasingly being managed by implantable devices. The patient population chosen for the first human trials might be patients with implanted ventricular assist devices (VADs), in which the indwelling pumps remove load from the heart and preserve blood flow despite poor myocardial function and arrhythmic events. Interestingly, the presence of VADs can result in some improvement of the host myocardium.7Heerdt PM Holmes JW Cai B Barbone A Madigan JD Reiken S et al.Chronic unloading by left ventricular assist device reverses contractile dysfunction and alters gene expression in end-stage heart failure.Circulation. 2001; 102: 2713-2719Crossref Scopus (256) Google Scholar,8Birks EJ Tansley PD Hardy J George RS Bowles CT Burke M et al.Left ventricular assist device and drug therapy for the reversal of heart failure.N Engl J Med. 2006; 355: 1873-1884Crossref PubMed Scopus (723) Google Scholar We might even think of the large cell grafts, as used in this study, as cellular VADs, replacing metal and plastic with living tissue and both providing new muscle and promoting recovery of the old and damaged heart.