Abstract
The application of tissue-engineering approaches to human induced pluripotent stem (hiPS) cells enables the development of physiologically relevant human tissue models for in vitro studies of development, regeneration, and disease. However, the immature phenotype of hiPS-derived cardiomyocytes (hiPS-CMs) limits their utility. We have developed a protocol to generate engineered cardiac tissues from hiPS cells and electromechanically mature them toward an adult-like phenotype. This protocol also provides optimized methods for analyzing these tissues' functionality, ultrastructure, and cellular properties. The approach relies on biological adaptation of cultured tissues subjected to biomimetic cues, applied at an increasing intensity, to drive accelerated maturation. hiPS cells are differentiated into cardiomyocytes and used immediately after the first contractions are observed, when they still have developmental plasticity. This starting cell population is combined with human dermal fibroblasts, encapsulated in a fibrin hydrogel and allowed to compact under passive tension in a custom-designed bioreactor. After 7 d of tissue formation, the engineered tissues are matured for an additional 21 d by increasingly intense electromechanical stimulation. Tissue properties can be evaluated by measuring contractile function, responsiveness to electrical stimuli, ultrastructure properties (sarcomere length, mitochondrial density, networks of transverse tubules), force-frequency and force-length relationships, calcium handling, and responses to β-adrenergic agonists. Cell properties can be evaluated by monitoring gene/protein expression, oxidative metabolism, and electrophysiology. The protocol takes 4 weeks and requires experience in advanced cell culture and machining methods for bioreactor fabrication. We anticipate that this protocol will improve modeling of cardiac diseases and testing of drugs.
Highlights
Advances in stem cell biology and tissue engineering have led to the development of engineered tissue models or “organs-on-a-chip”, intended to serve as physiologically relevant human in vitro models of their in vivo counterparts
The immature human induced pluripotent stem (hiPS) derived cardiomyocytes express the inward funny channel (If), which may cause arrhythmias when implanted into an adult heart [14]
We found that the cardiac differentiation protocols that use cell monolayers are more reproducible than those using embryoid bodies (EB), and that dissociation of cell monolayers is more robust and less damaging to the cells
Summary
Advances in stem cell biology and tissue engineering have led to the development of engineered tissue models or “organs-on-a-chip”, intended to serve as physiologically relevant human in vitro models of their in vivo counterparts. Matured engineered cardiac tissues were formed from early-stage hiPS-CM cells 10–12 days after the beginning of differentiation (Figure 2A). Cell alignment and force generation lead to physiological hypertrophy, with post-natal increases in cardiomyocytes mass and establishing a sarcomere length optimized for force production [6] By mimicking this process in vitro at an increased level of electromechanical conditioning, and combining the best cardiac tissue engineering approaches of others in the field [2,3,4,5, 8], this protocol is able to mature the engineered cardiac tissues beyond currently achievable levels and reverse the characteristically negative force-frequency relationships seen in hiPS-CM.
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