Hans Reinecke

Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts

The mammalian heart has a very limited regenerative capacity and, hence, heals by scar formation. Recent reports suggest that haematopoietic stem cells can transdifferentiate into unexpected phenotypes such as skeletal muscle, hepatocytes, epithelial cells, neurons, endothelial cells and cardiomyocytes, in response to tissue injury or placement in a new environment. Furthermore, transplanted human hearts contain myocytes derived from extra-cardiac progenitor cells, which may have originated from bone marrow. Although most studies suggest that transdifferentiation is extremely rare under physiological conditions, extensive regeneration of myocardial infarcts was reported recently after direct stem cell injection, prompting several clinical trials. Here, we used both cardiomyocyte-restricted and ubiquitously expressed reporter transgenes to track the fate of haematopoietic stem cells after 145 transplants into normal and injured adult mouse hearts. No transdifferentiation into cardiomyocytes was detectable when using these genetic techniques to follow cell fate, and stem-cell-engrafted hearts showed no overt increase in cardiomyocytes compared to sham-engrafted hearts. These results indicate that haematopoietic stem cells do not readily acquire a cardiac phenotype, and raise a cautionary note for clinical studies of infarct repair.

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)

Survival, Integration, and Differentiation of Cardiomyocyte Grafts A Study in Normal and Injured Rat Hearts

BACKGROUND Cardiomyocyte grafting augments myocyte numbers in the heart. We investigated (1) how developmental stage influences graft survival; (2) whether acutely necrotic or healing cardiac lesions support grafts; and (3) the differentiation and integration of cardiomyocyte grafts in injured hearts. METHODS AND RESULTS Cardiomyocytes from fetal, neonatal, or adult inbred rats were grafted into normal myocardium, acutely cryoinjured myocardium, or granulation tissue (6 days after injury). Adult cardiomyocytes did not survive under any conditions. In contrast, fetal and neonatal cardiomyocytes formed viable grafts under all conditions. Time-course studies with neonatal cardiomyocytes showed that the grafts recapitulated many aspects of normal development. The adherens junction protein N-cadherin was distributed circumferentially at day 1 but began to organize into intercalated disk-like structures by day 6. The gap junction protein connexin43 followed a similar but delayed pattern relative to N-cadherin. From 2 to 8 weeks, there was progressive hypertrophy and the formation of mature intercalated disks. In some hearts, graft cells formed adherens and gap junctions with host cardiomyocytes, suggesting electromechanical coupling. More commonly, however, grafts were separated from the host myocardium by scar tissue. Gap and adherens junctions formed between neonatal and adult cardiomyocytes in coculture, as evidenced by dye transfer and localization of cadherin and connexin43 at intercellular junctions. CONCLUSIONS Grafted fetal and neonatal cardiomyocytes form new, mature myocardium with the capacity to couple with injured host myocardium. Optimal repair, however, may require reducing the isolation of the graft by the intervening scar tissue.

Biphasic role for Wnt/β-catenin signaling in cardiac specification in zebrafish and embryonic stem cells

Understanding pathways controlling cardiac development may offer insights that are useful for stem cell-based cardiac repair. Developmental studies indicate that the Wnt/β-catenin pathway negatively regulates cardiac differentiation, whereas studies with pluripotent embryonal carcinoma cells suggest that this pathway promotes cardiogenesis. This apparent contradiction led us to hypothesize that Wnt/β-catenin signaling acts biphasically, either promoting or inhibiting cardiogenesis depending on timing. We used inducible promoters to activate or repress Wnt/β-catenin signaling in zebrafish embryos at different times of development. We found that Wnt/β-catenin signaling before gastrulation promotes cardiac differentiation, whereas signaling during gastrulation inhibits heart formation. Early treatment of differentiating mouse embryonic stem (ES) cells with Wnt-3A stimulates mesoderm induction, activates a feedback loop that subsequently represses the Wnt pathway, and increases cardiac differentiation. Conversely, late activation of β-catenin signaling reduces cardiac differentiation in ES cells. Finally, constitutive overexpression of the β-catenin-independent ligand Wnt-11 increases cardiogenesis in differentiating mouse ES cells. Thus, Wnt/β-catenin signaling promotes cardiac differentiation at early developmental stages and inhibits it later. Control of this pathway may promote derivation of cardiomyocytes for basic research and cell therapy applications.

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.

Growth of Engineered Human Myocardium With Mechanical Loading and Vascular Coculture

Rationale: The developing heart requires both mechanical load and vascularization to reach its proper size, yet the regulation of human heart growth by these processes is poorly understood. Objective: We seek to elucidate the responses of immature human myocardium to mechanical load and vascularization using tissue engineering approaches. Methods and Results: Using human embryonic stem cell and human induced pluripotent stem cell–derived cardiomyocytes in a 3-dimensional collagen matrix, we show that uniaxial mechanical stress conditioning promotes 2-fold increases in cardiomyocyte and matrix fiber alignment and enhances myofibrillogenesis and sarcomeric banding. Furthermore, cyclic stress conditioning markedly increases cardiomyocyte hypertrophy (2.2-fold) and proliferation rates (21%) versus unconditioned constructs. Addition of endothelial cells enhances cardiomyocyte proliferation under all stress conditions (14% to 19%), and addition of stromal supporting cells enhances formation of vessel-like structures by ≈10-fold. Furthermore, these optimized human cardiac tissue constructs generate Starling curves, increasing their active force in response to increased resting length. When transplanted onto hearts of athymic rats, the human myocardium survives and forms grafts closely apposed to host myocardium. The grafts contain human microvessels that are perfused by the host coronary circulation. Conclusions: Our results indicate that both mechanical load and vascular cell coculture control cardiomyocyte proliferation, and that mechanical load further controls the hypertrophy and architecture of engineered human myocardium. Such constructs may be useful for studying human cardiac development as well as for regenerative therapy.

Physiological function and transplantation of scaffold-free and vascularized human cardiac muscle tissue

Success of human myocardial tissue engineering for cardiac repair has been limited by adverse effects of scaffold materials, necrosis at the tissue core, and poor survival after transplantation due to ischemic injury. Here, we report the development of scaffold-free prevascularized human heart tissue that survives in vivo transplantation and integrates with the host coronary circulation. Human embryonic stem cells (hESCs) were differentiated to cardiomyocytes by using activin A and BMP-4 and then placed into suspension on a rotating orbital shaker to create human cardiac tissue patches. Optimization of patch culture medium significantly increased cardiomyocyte viability in patch centers. These patches, composed only of enriched cardiomyocytes, did not survive to form significant grafts after implantation in vivo. To test the hypothesis that ischemic injury after transplantation would be attenuated by accelerated angiogenesis, we created “second-generation,” prevascularized, and entirely human patches from cardiomyocytes, endothelial cells (both human umbilical vein and hESC-derived endothelial cells), and fibroblasts. Functionally, vascularized patches actively contracted, could be electrically paced, and exhibited passive mechanics more similar to myocardium than patches comprising only cardiomyocytes. Implantation of these patches resulted in 10-fold larger cell grafts compared with patches composed only of cardiomyocytes. Moreover, the preformed human microvessels anastomosed with the rat host coronary circulation and delivered blood to the grafts. Thus, inclusion of vascular and stromal elements enhanced the in vitro performance of engineered human myocardium and markedly improved viability after transplantation. These studies demonstrate the importance of including vascular and stromal elements when designing human tissues for regenerative therapies.

Systems approaches to preventing transplanted cell death in cardiac repair

Stem cell transplantation may repair the injured heart, but tissue regeneration is limited by death of transplanted cells. Most cell death occurs in the first few days post-transplantation, likely from a combination of ischemia, anoikis and inflammation. Interventions known to enhance transplanted cell survival include heat shock, over-expressing anti-apoptotic proteins, free radical scavengers, anti-inflammatory therapy and co-delivery of extracellular matrix molecules. Combinatorial use of such interventions markedly enhances graft cell survival, but death still remains a significant problem. We review these challenges to cardiac cell transplantation and present an approach to systematically address them. Most anti-death studies use histology to assess engraftment, which is time- and labor-intensive. To increase throughput, we developed two biochemical approaches to follow graft viability in the mouse heart. The first relies on LacZ enzymatic activity to track genetically modified cells, and the second quantifies human genomic DNA content using repetitive Alu sequences. Both show linear relationships between input cell number and biochemical signal, but require correction for the time lag between cell death and loss of signal. Once optimized, they permit detection of as few as 1 graft cell in 40,000 host cells. Pro-survival effects measured biochemically at three days predict long-term histological engraftment benefits. These methods permitted identification of carbamylated erythropoietin (CEPO) as a pro-survival factor for human embryonic stem cell-derived cardiomyocyte grafts. CEPOs effects were additive to heat shock, implying independent survival pathways. This system should permit combinatorial approaches to enhance graft viability in a fraction of the time required for conventional histology.