Pluripotent stem cells provide a potential solution to current epidemic rates of heart failure 1 by providing human cardiomyocytes to support heart regeneration 2. Studies of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) in small animal models have shown favorable effects of this treatment 3–7. It remains unknown, however, whether clinical scale hESC-CMs transplantation is feasible, safe or can provide large-scale myocardial regeneration. Here we show that hESC-CMs can be produced at a clinical scale (>1 billion cells/batch) and cryopreserved with good viability. Using a non-human primate (NHP) model of myocardial ischemia-reperfusion, we show that that cryopreservation and intra-myocardial delivery of 1 billion hESC-CMs generates significant remuscularization of the infarcted heart. The hESC-CMs showed progressive but incomplete maturation over a three-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 7, 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.

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 three 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 strain markedly increases cardiomyocyte hypertrophy (2.2-fold) and proliferation rates (21%) vs. unstrained 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 co-culture 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.

The discovery of human pluripotent stem cells (hPSCs), including both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), has opened up novel paths for a wide range of scientific studies. The capability to direct the differentiation of hPSCs into functional cardiomyocytes has provided a platform for regenerative medicine, development, tissue engineering, disease modeling, and drug toxicity testing. Despite exciting progress, achieving the optimal benefits has been hampered by the immature nature of these cardiomyocytes. Cardiac maturation has long been studied in vivo using animal models, but finding ways to mature hPSC cardiomyocytes (hPSC-CMs) is only in its initial stages. In this review, we discuss progress in promoting the maturation of the hPSC-CMs, in the context of our current knowledge of developmental cardiac maturation and in relation to in vitro model systems such as rodent ventricular myocytes. Promising approaches that have begun to be examined in hPSC-CMs include long-term culturing, three dimensional tissue engineering, mechanical loading, electrical stimulation, modulation of substrate stiffness and treatment with neurohormonal factors. Future studies will benefit from the combinatorial use of different approaches that more closely mimic nature’s diverse cues, which may result in broader changes in structure, function, and therapeutic applicability.

Understanding pathways controlling cardiac development may offer insights that are useful for stem cell-based cardiac repair. Developmental studies indicate that the Wnt/␤-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/␤-catenin signaling acts biphasically, either promoting or inhibiting cardiogenesis depending on timing. We used inducible promoters to activate or repress Wnt/␤-catenin signaling in zebrafish embryos at different times of development. We found that Wnt/␤-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 ␤-catenin signaling reduces cardiac differentiation in ES cells. Finally, constitutive overexpression of the ␤-catenin-independent ligand Wnt-11 increases cardiogenesis in differentiating mouse ES cells. Thus, Wnt/␤-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.heart development ͉ mesoderm ͉ Dickkopf-1 ͉ regeneration