Michael A. Laflamme

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.

Regenerating the heart

Cell-based cardiac repair offers the promise of rebuilding the injured heart from its component parts. Work began with committed cells such as skeletal myoblasts, but recently the field has expanded to explore an array of cell types, including bone marrow cells, endothelial progenitors, mesenchymal stem cells, resident cardiac stem cells, and both mouse and human embryonic stem cells. A related strategy for cardiac repair involves cell mobilization with factors such as cytokines. Translation of cell-based approaches to the clinic has progressed rapidly, and clinical trials using autologous skeletal myoblasts and bone marrow cells are under way. Many challenges remain before the vision of healing an infarct by muscle regeneration can be realized. Future research is likely to focus on improving our ability to guide the differentiation of stem cells, control their survival and proliferation, identify factors that mediate their homing and modulate the hearts innate inflammatory and fibrotic responses.

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)

Human ES-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts

Transplantation studies in mice and rats have shown that human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) can improve the function of infarcted hearts, but two critical issues related to their electrophysiological behaviour in vivo remain unresolved. First, the risk of arrhythmias following hESC-CM transplantation in injured hearts has not been determined. Second, the electromechanical integration of hESC-CMs in injured hearts has not been demonstrated, so it is unclear whether these cells improve contractile function directly through addition of new force-generating units. Here we use a guinea-pig model to show that hESC-CM grafts in injured hearts protect against arrhythmias and can contract synchronously with host muscle. Injured hearts with hESC-CM grafts show improved mechanical function and a significantly reduced incidence of both spontaneous and induced ventricular tachycardia. To assess the activity of hESC-CM grafts in vivo, we transplanted hESC-CMs expressing the genetically encoded calcium sensor, GCaMP3 (refs 4, 5). By correlating the GCaMP3 fluorescent signal with the host ECG, we found that grafts in uninjured hearts have consistent 1:1 host–graft coupling. Grafts in injured hearts are more heterogeneous and typically include both coupled and uncoupled regions. Thus, human myocardial grafts meet physiological criteria for true heart regeneration, providing support for the continued development of hESC-based cardiac therapies for both mechanical and electrical repair.

Evidence for Cardiomyocyte Repopulation by Extracardiac Progenitors in Transplanted Human Hearts

Human myocardium has long been considered to have essentially no intrinsic regenerative capacity. Recent studies in rodent models, however, have suggested the presence of an extracardiac stem cell population, perhaps in bone marrow, that is capable of some reconstitution of cardiomyocytes after injury. To determine whether similar mechanisms exist in the human heart, we evaluated human female allograft hearts transplanted into male patients. The presence of Y chromosomes in cardiomyocytes would indicate these cells arose from the recipient, rather than the donor heart. We identified 5 male patients who had retained a female heart at least 9 months before death and necropsy. Remarkably, in each case, the transplanted heart contained a minute but readily detectable fraction of Y chromosome-positive cardiomyocytes. The mean percentage of cardiomyocytes arising from the host was estimated to be 0.04% with a median of 0.016%. Most Y-positive cardiomyocytes were associated with regions of acute rejection, suggesting such chimerism involves an injury event. Furthermore, the sole patient whose immediate cause of death was allograft rejection showed a much higher percentage of host-derived cardiomyocytes, up to 29% in local, 1-mm2 “hot spots.” Thus, adult humans have extracardiac progenitor cells capable of migrating to and repopulating damaged myocardium, but this process occurs at very low levels.

Formation of Human Myocardium in the Rat Heart from Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) offer the opportunity to replenish cells lost in the postinfarct heart. We explored whether human myocardium could be generated in rat hearts by injecting differentiated cardiac-enriched hESC progeny into the left ventricular wall of athymic rats. Although initial grafts were predominantly epithelial, noncardiac elements were lost over time, and grafts consisted predominantly of cardiomyocytes by 4 weeks. No teratomatous elements were observed. Engrafted cardiomyocytes were glycogen-rich and expressed expected cardiac markers including beta-myosin heavy chain, myosin light chain 2v, and atrial natriuretic factor. Heat-shock treatment improved graft size approximately threefold. The cardiac implants exhibited substantial angiogenesis, both recipient and graft derived. Importantly, there was greater proliferation in human cardiomyocytes than previously seen in rodent-derived cardiomyocytes: 14.4% of graft cardiomyocytes expressed the proliferation marker Ki-67, and 2.7% incorporated the thymidine analog BrdU 4 weeks after transplantation. This proliferation was associated with a sevenfold increase in graft size over the 4-week interval. Thus, hESCs can form human myocardium in the rat heart, permitting studies of human myocardial development and physiology and supporting the feasibility of their use in myocardial repair.

Proangiogenic scaffolds as functional templates for cardiac tissue engineering

We demonstrate here a cardiac tissue-engineering strategy addressing multicellular organization, integration into host myocardium, and directional cues to reconstruct the functional architecture of heart muscle. Microtemplating is used to shape poly(2-hydroxyethyl methacrylate-co-methacrylic acid) hydrogel into a tissue-engineering scaffold with architectures driving heart tissue integration. The construct contains parallel channels to organize cardiomyocyte bundles, supported by micrometer-sized, spherical, interconnected pores that enhance angiogenesis while reducing scarring. Surface-modified scaffolds were seeded with human ES cell-derived cardiomyocytes and cultured in vitro. Cardiomyocytes survived and proliferated for 2 wk in scaffolds, reaching adult heart densities. Cardiac implantation of acellular scaffolds with pore diameters of 30–40 μm showed angiogenesis and reduced fibrotic response, coinciding with a shift in macrophage phenotype toward the M2 state. This work establishes a foundation for spatially controlled cardiac tissue engineering by providing discrete compartments for cardiomyocytes and stroma in a scaffold that enhances vascularization and integration while controlling the inflammatory response.

Biowire: a platform for maturation of human pluripotent stem cell–derived cardiomyocytes

Directed differentiation protocols enable derivation of cardiomyocytes from human pluripotent stem cells (hPSCs) and permit engineering of human myocardium in vitro. However, hPSC-derived cardiomyocytes are reflective of very early human development, limiting their utility in the generation of in vitro models of mature myocardium. Here we describe a platform that combines three-dimensional cell cultivation with electrical stimulation to mature hPSC-derived cardiac tissues. We used quantitative structural, molecular and electrophysiological analyses to explain the responses of immature human myocardium to electrical stimulation and pacing. We demonstrated that the engineered platform allows for the generation of three-dimensional, aligned cardiac tissues (biowires) with frequent striations. Biowires submitted to electrical stimulation had markedly increased myofibril ultrastructural organization, elevated conduction velocity and improved both electrophysiological and Ca2+ handling properties compared to nonstimulated controls. These changes were in agreement with cardiomyocyte maturation and were dependent on the stimulation rate.

Structural and Functional Maturation of Cardiomyocytes Derived from Human Pluripotent Stem Cells

Despite preclinical studies demonstrating the functional benefit of transplanting human pluripotent stem cell-derived cardiomyocytes (PSC-CMs) into damaged myocardium, the ability of these immature cells to adopt a more adult-like cardiomyocyte (CM) phenotype remains uncertain. To address this issue, we tested the hypothesis that prolonged in vitro culture of human embryonic stem cell (hESC)- and human induced pluripotent stem cell (hiPSC)-derived CMs would result in the maturation of their structural and contractile properties to a more adult-like phenotype. Compared to their early-stage counterparts (PSC-CMs after 20-40 days of in vitro differentiation and culture), late-stage hESC-CMs and hiPSC-CMs (80-120 days) showed dramatic differences in morphology, including increased cell size and anisotropy, greater myofibril density and alignment, sarcomeres visible by bright-field microscopy, and a 10-fold increase in the fraction of multinucleated CMs. Ultrastructural analysis confirmed improvements in the myofibrillar density, alignment, and morphology. We measured the contractile performance of late-stage hESC-CMs and hiPSC-CMs and noted a doubling in shortening magnitude with slowed contraction kinetics compared to the early-stage cells. We then examined changes in the calcium-handling properties of these matured CMs and found an increase in calcium release and reuptake rates with no change in the maximum amplitude. Finally, we performed electrophysiological assessments in hESC-CMs and found that late-stage myocytes have hyperpolarized maximum diastolic potentials, increased action potential amplitudes, and faster upstroke velocities. To correlate these functional changes with gene expression, we performed qPCR and found a robust induction of the key cardiac structural markers, including β-myosin heavy chain and connexin-43, in late-stage hESC-CMs and hiPSC-CMs. These findings suggest that PSC-CMs are capable of slowly maturing to more closely resemble the phenotype of adult CMs and may eventually possess the potential to regenerate the lost myocardium with robust de novo force-producing tissue.