Spontaneous formation of extensive vessel-like structures in murine engineered heart tissue.

Engineered heart tissue (EHT) from primary heart cells contains endothelial cells (ECs), but the extent to which ECs organize into vessel-like structures or even functional vessels remains unknown and is difficult to study by conventional methods. In this study, we generated fibrin-based mini-EHTs from a transgenic mouse line (Cdh5-CreERT2 x Rosa26-LacZ), in which ECs were specifically and inducibly labeled by applying tamoxifen (ECiLacZ). EHTs were generated from an unpurified cell mix of newborn mouse hearts and were cultured under standard serum-containing conditions. Cre expression in 15-day-old EHTs was induced by addition of o-hydroxytamoxifen to the culture medium for 48h, and ECs were visualized by X-gal staining. ECiLacZ EHTs showed a dense X-gal-positive vessel-like network with distinct tubular structures. Immunofluorescence revealed that ECs were mainly associated with cardiomyocytes within the EHT. ECiLacZ EHT developed spontaneous and regular contractility with forces up to 0.1mN. Coherent contractility and the presence of an extensive vessel-like network were both dependent on the presence of animal sera in the culture medium. Contractile ECiLacZ EHTs successfully served as grafts in implantation studies onto the hearts of immunodeficient mice. Four weeks after implantation, EHTs showed X-gal-positive lumen-forming vessel structures connected to the host myocardium circulation as they contained erythrocytes on a regular basis. Taken together, genetic labeling of ECs revealed the extensive formation of vessel-like structures in EHTs in vitro. The ECiLacZ EHT model could help simultaneously study biological effects of compounds on cardiomyocyte function and tissue vascularization.