Elina Minami

Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts

Cardiomyocytes derived from human embryonic stem (hES) cells potentially offer large numbers of cells to facilitate repair of the infarcted heart. However, this approach has been limited by inefficient differentiation of hES cells into cardiomyocytes, insufficient purity of cardiomyocyte preparations and poor survival of hES cell–derived myocytes after transplantation. Seeking to overcome these challenges, we generated highly purified human cardiomyocytes using a readily scalable system for directed differentiation that relies on activin A and BMP4. We then identified a cocktail of pro-survival factors that limits cardiomyocyte death after transplantation. These techniques enabled consistent formation of myocardial grafts in the infarcted rat heart. The engrafted human myocardium attenuated ventricular dilation and preserved regional and global contractile function after myocardial infarction compared with controls receiving noncardiac hES cell derivatives or vehicle. The ability of hES cell–derived cardiomyocytes to partially remuscularize myocardial infarcts and attenuate heart failure encourages their study under conditions that closely match human disease.

Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts

Pluripotent stem cells provide a potential solution to current epidemic rates of heart failure by providing human cardiomyocytes to support heart regeneration. Studies of human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) in small-animal models have shown favourable effects of this treatment. However, it remains unknown whether clinical-scale hESC-CM transplantation is feasible, safe or can provide sufficient myocardial regeneration. Here we show that hESC-CMs can be produced at a clinical scale (more than one billion cells per batch) and cryopreserved with good viability. Using a non-human primate model of myocardial ischaemia followed by reperfusion, we show that cryopreservation and intra-myocardial delivery of one billion hESC-CMs generates extensive remuscularization of the infarcted heart. The hESC-CMs showed progressive but incomplete maturation over a 3-month period. Grafts were perfused by host vasculature, and electromechanical junctions between graft and host myocytes were present within 2 weeks of engraftment. Importantly, grafts showed regular calcium transients that were synchronized to the host electrocardiogram, indicating electromechanical coupling. In contrast to small-animal models, non-fatal ventricular arrhythmias were observed in hESC-CM-engrafted primates. Thus, hESC-CMs can remuscularize substantial amounts of the infarcted monkey heart. Comparable remuscularization of a human heart should be possible, but potential arrhythmic complications need to be overcome.

Transplantation of undifferentiated murine embryonic stem cells in the heart: teratoma formation and immune response

Embryonic stem (ES) cells are promising for cardiac repair’ but directing their differentiation toward cardiomyocytes remains challenging. We investigated whether the heart guides ES cells toward cardiomyocytes in vivo and whether allogeneic ES cells were immunologically tolerated. Undifferentiated mouse ES cells consistently formed cardiac teratomas in nude or immunocompetent syngeneic mice. Cardiac teratomas contained no more cardiomyocytes than hind‐limb teratomas’ suggesting lack of guided differentiation. ES cells also formed teratomas in infarcted hearts’ indicating injury‐related signals did not direct cardiac differentiation. Allogeneic ES cells also caused cardiac teratomas’ but these were immunologically rejected after several weeks’ in association with increased inflammation and up‐regulation of class I and II histocompatibility antigens. Fusion between ES cells and cardiomyocytes occurred in vivo’ but was rare. Infarct autofluorescence was identified as an artifact that might be mistaken for enhanced GFP expression and true regeneration. Hence’ undifferentiated ES cells were not guided toward a cardiomyocyte fate in either normal or infarcted hearts’ and there was no evidence for allogeneic immune tolerance of ES cell derivatives. Successful cardiac repair strategies involving ES cells will need to control cardiac differentiation’ avoid introducing undifferentiated cells’ and will likely require immune modulation to avoid rejection.—Nussbaum, J., Minami, E., Laflamme, M. A., Virag, J. A. I., Ware, C. B., Masino, A., Muskheli, V., Pabon, L., Reinecke, H., Murry, C. E. Transplantation of undifferentiated mu‐rine embryonic stem cells in the heart: teratoma formation and immune response. FASEB J. 21, 1345–1357 (2007)

rAAV6-microdystrophin preserves muscle function and extends lifespan in severely dystrophic mice.

Mice carrying mutations in both the dystrophin and utrophin genes die prematurely as a consequence of severe muscular dystrophy. Here, we show that intravascular administration of recombinant adeno-associated viral (rAAV) vectors carrying a microdystrophin gene restores expression of dystrophin in the respiratory, cardiac and limb musculature of these mice, considerably reducing skeletal muscle pathology and extending lifespan. These findings suggest rAAV vector–mediated systemic gene transfer may be useful for treatment of serious neuromuscular disorders such as Duchenne muscular dystrophy.

Evidence for Fusion Between Cardiac and Skeletal Muscle Cells

Abstract— Cardiomyoplasty with skeletal myoblasts may benefit cardiac function after infarction. Recent reports indicate that adult stem cells can fuse with other cell types. Because myoblasts are “fusigenic” cells by nature, we hypothesized they might be particularly likely to fuse with cardiomyocytes. To test this, neonatal rat cardiomyocytes labeled with LacZ and green fluorescent protein (GFP) were cocultured with unlabeled C2C12 myoblasts. After 3 days, we observed a small population of skeletal myotubes that expressed LacZ and GFP, indicating cell fusion. To test whether such fusion occurred in vivo, LacZ-expressing C2C12 myoblasts were grafted into normal nude mouse hearts. At 2 weeks after grafting, cells at the graft-host interface expressed both LacZ and cardiac-specific myosin light chain 2v (MLC2v). To test more definitively whether fusion between skeletal and cardiac muscle could occur, we used a Cre/lox reporter system that activated LacZ only upon cell fusion. When neonatal cardiomyocytes from &agr;-myosin heavy chain promoter (&agr;-MHC)-Cre mice were cocultured with myoblasts from floxed-lacZ reporter mice, LacZ was activated in a subset of cells, indicating cell fusion occurred in vitro. Finally, we grafted the floxed-lacZ myoblasts into normal hearts of &agr;-MHC-Cre+ and &agr;-MHC-Cre− mice (n=5 each). Hearts analyzed at 4 days and 1 week after transplantation demonstrated activation of LacZ when the skeletal muscle cells were implanted into hearts of &agr;-MHC-Cre+ mice, but not after implantation into &agr;-MHC-Cre− mice. These data indicate that skeletal muscle cell grafting gives rise to a subpopulation of skeletal-cardiac hybrid cells with a currently unknown phenotype. The full text of this article is available online at http://circres.ahajournals.org.

Cardiogenic Differentiation and Transdifferentiation of Progenitor Cells

In recent years, cell transplantation has drawn tremendous interest as a novel approach to preserving or even restoring contractile function to infarcted hearts. A typical human infarct involves the loss of approximately 1 billion cardiomyocytes, and, therefore, many investigators have sought to identify endogenous or exogenous stem cells with the capacity to differentiate into committed cardiomyocytes and repopulate lost myocardium. As a result of these efforts, dozens of stem cell types have been reported to have cardiac potential. These include pluripotent embryonic stem cells, as well various adult stem cells resident in compartments including bone marrow, peripheral tissues, and the heart itself. Some of these cardiogenic progenitors have been reported to contribute replacement muscle through endogenous reparative processes or via cell transplantation in preclinical cardiac injury models. However, considerable disagreement exists regarding the efficiency and even the reality of cardiac differentiation by many of these stem cell types, making these issues a continuing source of controversy in the field. In this review, we consider approaches to cell fate mapping and establishing the cardiac phenotype, as well as the present state of the evidence for the cardiogenic and regenerative potential of the major candidate stem cell types.

Sildenafil reverses cardiac dysfunction in the mdx mouse model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a progressive and fatal genetic disorder of muscle degeneration. Patients with DMD lack expression of the protein dystrophin as a result of mutations in the X-linked dystrophin gene. The loss of dystrophin leads to severe skeletal muscle pathologies as well as cardiomyopathy, which manifests as congestive heart failure and arrhythmias. Like humans, dystrophin-deficient mice (mdx mice) show cardiac dysfunction as evidenced by a decrease in diastolic function followed by systolic dysfunction later in life. We have investigated whether sildenafil citrate (Viagra), a phosphodiesterase 5 (PDE5) inhibitor, can be used to ameliorate the age-related cardiac dysfunction present in the mdx mice. By using echocardiography, we show that chronic sildenafil treatment reduces functional deficits in the cardiac performance of aged mdx mice, with no effect on normal cardiac function in WT controls. More importantly, when sildenafil treatment was started after cardiomyopathy had developed, the established symptoms were rapidly reversed within a few days. It is recognized that PDE5 inhibitors can have cardioprotective effects in other models of cardiac damage, but the present study reports a prevention and reversal of pathological cardiac dysfunction as measured by functional analysis in a mouse model of DMD. Overall, the data suggest that PDE5 inhibitors may be a useful treatment for the cardiomyopathy affecting patients with DMD at early and late stages of the disease.

NFATc3-Induced Reductions in Voltage-Gated K+ Currents After Myocardial Infarction

Reductions in voltage-activated K+ (Kv) currents may underlie arrhythmias after myocardial infarction (MI). We investigated the role of β-adrenergic signaling and the calcineurin/NFAT pathway in mediating the reductions in Kv currents observed after MI in mouse ventricular myocytes. Kv currents were produced by the summation of 3 distinct currents: Ito, IKslow1, and IKslow2. At 48 hours after MI, we found a 4-fold increase in NFAT activity, which coincided with a decrease in the amplitudes of Ito, IKslow1, and IKslow2. Consistent with this, mRNA and protein levels of Kv1.5, 2.1, 4.2, and 4.3, which underlie IKslow1, IKslow2, and Ito, were decreased after MI. Administration of the β-blocker metoprolol prevented the activation of NFAT and the reductions in Ito, IKslow1, and IKslow2 after MI. Cyclosporine, an inhibitor of calcineurin, also prevented the reductions in these currents after MI. Importantly, Kv currents did not change after MI in ventricular myocytes from NFATc3 knockout mice. Conversely, chronic β-adrenergic stimulation or expression of an activated NFATc3 decreased Kv currents to a similar extent as MI. Taken together, these data indicate that NFATc3 plays an essential role in the signaling pathway leading to reduced Ito, IKslow1, and IKslow2 after MI. We propose that increased β-adrenergic signaling after MI activates calcineurin and NFATc3, which decreases Ito, IKslow1, and IKslow2 via a reduction in Kv1.5, Kv2.1, Kv4.2, and Kv4.3 expression.

Skeletal muscle meets cardiac muscle. Friends or foes

Since the early 1990s, cellular cardiomyoplasty (or simply cell transplantation) has been explored as a potential new therapy to alleviate the consequences of myocardial infarction (MI) [(1)][1]. Many different cell types have been tried [(2)][2]and, quite naturally, cardiomyocytes were chosen early

Electrical integration of human embryonic stem cell-derived cardiomyocytes in a guinea pig chronic infarct model

Background: Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) were recently shown to be capable of electromechanical integration following direct injection into intact or recently injured guinea pig hearts, and hESC-CM transplantation in recently injured hearts correlated with improvements in contractile function and a reduction in the incidence of arrhythmias. The present study was aimed at determining the ability of hESC-CMs to integrate and modulate electrical stability following transplantation in a chronic model of cardiac injury. Methods and Results: At 28 days following cardiac cryoinjury, guinea pigs underwent intracardiac injection of hESC-CMs, noncardiac hESC derivatives (non-CMs), or vehicle. Histology confirmed partial remuscularization of the infarct zone in hESC-CM recipients while non-CM recipients showed heterogeneous xenografts. The 3 experimental groups showed no significant difference in the left ventricular dimensions or fractional shortening by echocardiography or in the incidence of spontaneous arrhythmias by telemetric monitoring. Although recipients of hESC-CMs and vehicle showed a similar incidence of arrhythmias induced by programmed electrical stimulation at 4 weeks posttransplantation, non-CM recipients proved to be highly inducible, with a ∼3-fold greater incidence of induced arrhythmias. In parallel studies, we investigated the ability of hESC-CMs to couple with host myocardium in chronically injured hearts by the intravital imaging of hESC-CM grafts that stably expressed a fluorescent reporter of graft activation, the genetically encoded calcium sensor GCaMP3. In this work, we found that only ∼38% (5 of 13) of recipients of GCaMP3+ hESC-CMs showed fluorescent transients that were coupled to the host electrocardiogram. Conclusions: Human embryonic stem cell-derived cardiomyocytes engraft in chronically injured hearts without increasing the incidence of arrhythmias, but their electromechanical integration is more limited than previously reported following their transplantation in a subacute injury model. Moreover, non-CM grafts may promote arrhythmias under certain conditions, a finding that underscores the need for input preparations of high cardiac purity.