Natural polymer hydrogels are used ubiquitously as scaffold materials for cardiac tissue engineering as well as for soft tissue engineering more broadly due to FDA approval, minimal immunogenicity, and well defined physiological clearance pathways. However, the relationships between natural polymer hydrogels and resident cell populations in directing the development of engineered tissues are poorly defined. This is of particular concern for tissues prepared with iPSC-derived cell populations, in which population purity and batch-to-batch variability become additional critical factors to consider. Herein, the design space for a blended fibrin and collagen scaffold is characterized for applications in creating engineered myocardium with human iPSC-derived cardiomyocytes. Stiffness values of the acellular hydrogel formulations considered approach those of native myocardium in compression, but deviate significantly in tension when compared to rat myocardium in both transverse and longitudinal fiber orientations. A response surface methodology approach to understanding the relationship between collagen concentration, fibrin concentration, seeding density, and cardiac purity found a statistically significant predictive model across three repeated studies that confirms that all of these factors contribute to tissue compaction. In these constructs, increased fibrin concentration and seeding density were each associated with increased compaction, while increased collagen concentration was associated with decreased compaction. Both the lowest (24.4% cTnT⁺) and highest (60.2% cTnT⁺) cardiomyocyte purities evaluated were associated with decreased compaction, while the greatest compaction was predicted to occur in constructs prepared with a 40-50% cTnT⁺ population. Constructs prepared with purified cardiomyocytes (≥75.5% cTnT⁺) compacted and formed syncytia well, although increased fibrin concentration in these groups was associated with decreased compaction, a reversal of the trend observed in unpurified cardiomyocytes. This study demonstrates an analytical approach to understanding cell-scaffold interactions in engineered tissues and provides a foundation for the development of more sophisticated and customized scaffold platforms for human cardiac tissue engineering.

Cardiac tissue engineering is a promising approach to provide large-scale tissues for transplantation to regenerate the heart after ischemic injury, however, integration with the host myocardium will be required to achieve electromechanical benefits. To test the ability of engineered heart tissues to electrically integrate with the host, 10 million human embryonic stem cell (hESC)-derived cardiomyocytes were used to form either scaffold-free tissue patches implanted on the epicardium or micro-tissue particles (~1000 cells/particle) delivered by intramyocardial injection into the left ventricular wall of the ischemia/reperfusion injured athymic rat heart. Results were compared to intramyocardial injection of 10 million dispersed hESC-cardiomyocytes. Graft size was not significantly different between treatment groups and correlated inversely with infarct size. After implantation on the epicardial surface, hESC-cardiac tissue patches were electromechanically active, but they beat slowly and were not electrically coupled to the host at 4 weeks based on ex vivo fluorescent imaging of their graft-autonomous GCaMP3 calcium reporter. Histologically, scar tissue physically separated the patch graft and host myocardium. In contrast, following intramyocardial injection of micro-tissue particles and suspended cardiomyocytes, 100% of the grafts detected by fluorescent GCaMP3 imaging were electrically coupled to the host heart at spontaneous rate and could follow host pacing up to a maximum of 300–390 beats per minute (5–6.5 Hz). Gap junctions between intramyocardial graft and host tissue were identified histologically. The extensive coupling and rapid response rate of the human myocardial grafts after intramyocardial delivery suggest electrophysiological adaptation of hESC-derived cardiomyocytes to the rat heart’s pacemaking activity. These data support the use of the rat model for studying electromechanical integration of human cardiomyocytes, and they identify lack of electrical integration as a challenge to overcome in tissue engineered patches.

Heart disease is the leading cause of morbidity and mortality worldwide, and regenerative therapies that replace damaged myocardium could benefit millions of patients annually. The many cell types in the heart, including cardiomyocytes, endothelial cells, vascular smooth muscle cells, pericytes, and cardiac fibroblasts, communicate via intercellular signaling and modulate each other's function. Although much progress has been made in generating cells of the cardiovascular lineage from human pluripotent stem cells, a major challenge now is creating the tissue architecture to integrate a microvascular circulation and afferent arterioles into such an engineered tissue. Recent advances in cardiac and vascular tissue engineering will move us closer to the goal of generating functionally mature tissue. Using the biology of the myocardium as the foundation for designing engineered tissue and addressing the challenges to implantation and integration, we can bridge the gap from bench to bedside for a clinically tractable engineered cardiac tissue.

Neuregulin-1 (NRG-1) and its signaling receptors, erythroblastic leukemia viral oncogene homologs (ErbB) 2, 3, and 4, have been implicated in both cardiomyocyte development and disease, as well as in homeostatic cardiac function. NRG-1/ErbB signaling is involved in a multitude of cardiac processes ranging from myocardial and cardiac conduction system development to angiogenic support of cardiomyocytes, to cardioprotective effects upon injury. Numerous studies of NRG-1 employ a variety of platforms, including in vitro assays, animal models, and human clinical trials, with equally varying and, sometimes, contradictory outcomes. NRG-1 has the potential to be used as a therapeutic tool in stem cell therapies, tissue engineering applications, and clinical diagnostics and treatment. This review presents a concise summary of the growing body of literature to highlight the temporally persistent significance of NRG-1/ErbB signaling throughout development, homeostasis, and disease in the heart, specifically in cardiomyocytes.