Abstract
Engineered heart tissue (EHT) is a 3D cardiac model made of hydrogel and cardiac cells cast in a mold anchored between two pillars. Since its introduction in 2002 (PMID: 11834716) it has become a key tool to reproduce the structure and function of native heart muscle in vitro. Developing multicellular EHTs (mEHTs) from human induced pluripotent stem cells (hiPSC)-derived cardiac cells provide a more physiological microenvironment, promoting cardiomyocyte (CM) maturation and generating tissues that better mimic the human myocardium (PMID: 35401829). This approach overcomes major limitations of current models, such as limited access to patient-derived CMs, low translational value of animal systems, and immaturity of 2D hiPSC-derived CMs.
Our goal is to establish a mEHT composed of hiPSC-derived CMs, endothelial cells (ECs) and cardiac fibroblasts (CFs): the three main cardiac cell types (PMID: 36443005). Using a healthy donor hiPSC line, CMs were obtained using a commercial differentiation media, while ECs and CFs were generated by optimizing a previously published differentiation protocol (PMID: 33772245) and validated by flow cytometry and/or immunofluorescence. EHTs were produced and analyzed using a commercial two-pillar device that enables culture, stimulation, and real-time contractility assessment via integrated optical fiber technology.
Contractile activity of mEHTs containing 70% CMs, 15% ECs, and 15% CFs was analyzed at day 7, 14 and 21. Preliminary results revealed significant functional maturation: beating rate decreased, while contractile force and contraction time increased between days 7–14 and 7–21, with no significant differences between days 14–21, indicating a stabilization phase. Relaxation time remained unchanged. Fluorescence imaging confirmed more elongated CMs and a compact, aligned tissue structure by day 14.
To further enhance physiological relevance, first attempts were made to integrate hiPSC-derived sympathetic neurons to obtain an innervated mEHT. Once validated, this system is expected to provide an advanced human cardiac platform for drug screening and disease modeling.
FUNDINGS: PROMOS (ITAT-11-35023 grant) Interreg VI-A Italia-Austria 2021-2027, co-funded by the European Union.