Background Tissue engineering enables the generation of functional human cardiac tissue using cells derived in vitro in combination with biocompatible materials. Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes provide a cell source for cardiac tissue engineering; however, their immaturity limits their potential applications. Here we sought to study the effect of mechanical conditioning and electrical pacing on the maturation of hiPSC-derived cardiac tissues. Methods Cardiomyocytes derived from hiPSCs were used to generate collagen-based bioengineered human cardiac tissue. Engineered tissue constructs were subjected to different mechanical stress and electrical pacing conditions. Results The engineered human myocardium exhibits Frank-Starling-type force-length relationships. After 2 weeks of static stress conditioning, the engineered myocardium demonstrated increases in contractility (0.63±0.10 mN/mm2 vs 0.055±0.009 mN/mm2 for no stress), tensile stiffness, construct alignment, and cell size. Stress conditioning also increased SERCA2 expression, which correlated with a less negative force-frequency relationship. When electrical pacing was combined with static stress conditioning, the tissues showed an additional increase in force production (1.34±0.19 mN/mm2), with no change in construct alignment or cell size, suggesting maturation of excitation-contraction coupling. Supporting this notion, we found expression of RYR2 and SERCA2 further increased by combined static stress and electrical stimulation. Conclusions These studies demonstrate that electrical pacing and mechanical stimulation promote maturation of the structural, mechanical and force generation properties of hiPSC-derived cardiac tissues.

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 enyzmatic 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. CEPO's 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.

Recent advances in pluripotent stem cell biology and directed differentiation have identified a population of human cardiovascular progenitors that give rise to cardiomyocytes, smooth muscle and endothelial cells. Because the heart develops from progenitors in 3-D under constant mechanical load, we sought to test the effects of a 3-D microenvironment and mechanical stress on differentiation and maturation of human cardiovascular progenitors into myocardial tissue. Progenitors were derived from embryonic stem cells, cast into collagen hydrogels, and left unstressed or subjected to static or cyclic mechanical stress. Compared to 2-D culture, the unstressed 3-D environment increased cardiomyocyte numbers and decreased smooth muscle numbers. Additionally, 3-D culture suppressed smooth muscle α-actin content, suggesting diminished cell maturation. Cyclic stress-conditioning increased expression of several cardiac markers, including β-myosin heavy chain and cardiac troponin T, and the tissue showed enhanced calcium dynamics and force production. There was no effect of mechanical loading on cardiomyocyte or smooth muscle specification. Thus, 3-D growth conditions favor cardiac differentiation from cardiovascular progenitors, whereas 2-D conditions promote smooth muscle differentiation. Mechanical loading promotes cardiomyocyte structural and functional maturation. Culture in 3-D facilitates understanding how cues such as mechanical stress affect the differentiation and morphogenesis of distinct cardiovascular cell populations into organized, functional human cardiovascular tissue.