Background
Understanding of human cardiac development and regeneration necessitates physiologically relevant models that accurately reflect multicellular interactions within cardiac tissue. Current in vitro approaches demonstrate limited complexity and cell-type diversity compared to native human cardiac tissue. Hence, we developed a multicellular bioengineered heart muscle (BHM) model derived from human induced pluripotent stem cells (hiPSCs) to reflect essential aspects of cardiac development, maturation, and stress-response mechanisms.
Methods
BHMs were generated through stage-specific directed differentiation of hiPSCs through three critical developmental phases: mesoderm induction (3 days), cardiac specification (10 days), and cardiac maturation (up to 60 days). Constructs were cast onto two flexible poles of distinct stiffnesses to evaluate mechano-maturational effects within 48-well plate format. Contractile function was assessed daily utilizing the Myr-Imager, a video-optical high-throughput imaging platform, quantifying fractional shortening, beating frequency, and contraction velocity as longitudinal functional readouts.
Results
BHMs exhibited robust contractile activity across differential stiffness conditions, demonstrating increased force generation that reached a functional plateau around day 30 with incremental increase until day 60. RNA sequencing analysis revealed progression through defined developmental stages that mirror human cardiac development. The key cardiac lineage transcription factors (NKX2-5, GATA4) and maturation markers (MYH7, TNNT2, TNNI3) demonstrated a sequential upregulation. Immunohistochemical characterization confirmed multicellular tissue complexity, predominantly comprising cardiomyocytes, fibroblast-like cells, endothelial cells, and WT1-positive epicardial cells. Advanced culture stages exhibited emergent of neuro-cardiac interfaces.
Conclusion
This multicellular bioengineered myocardial model successfully recapitulates critical developmental stages of human cardiac morphogenesis, functional maturation, and pathophysiological hypoxia-induced stress responses. This platform represents a powerful tool for investigating cardiac mechanobiology, modeling heart diseases, and developing novel regenerative therapies, with broad implications for translational cardiac research.
