RESUMO
OBJECTIVES: Univentricular malformations are severe cardiac lesions with limited therapeutic options and a poor long-term outcome. The staged surgical palliation (Fontan principle) results in a circulation in which venous return is conducted to the pulmonary arteries via passive laminar flow. We aimed to generate a contractile subpulmonary neo-ventricle from engineered heart tissue (EHT) to drive pulmonary flow actively. METHODS: A three-dimensional tubular EHT (1.8-cm length, 6-mm inner diameter, ca. 1-mm wall thickness) was created by casting human-induced pluripotent stem cell-derived cardiomyocytes (0.9 ml, 18 mio/ml) embedded in a fibrin-based hydrogel around a silicone tube. EHTs were cultured under continuous, pulsatile flow through the silicone tube for 23 days. RESULTS: The constructs started to beat macroscopically at days 8-14 and remained stable in size and shape over the whole culture period. Tubular EHTs showed a coherent beating pattern after 23 days in culture, and isovolumetric pressure measurements demonstrated a coherent pulsatile wave formation with an average frequency of 77 ± 5 beats/min and an average pressure of 0.2 mmHg. Histological analysis revealed cardiomyocytes mainly localized along the inner and outer curvature of the tubular wall with mainly longitudinal alignment. Cell density in the center of the tubular wall was lower. CONCLUSIONS: A simple tube-shaped contractile EHT was generated from human-induced pluripotent stem cells and developed a synchronous beating pattern. Further steps need to focus on optimizing support materials, flow rates and geometry to obtain a construct that creates sufficient pressures to support a directed and pulsatile blood flow.
Assuntos
Miócitos Cardíacos , Engenharia Tecidual , Fibrina , Ventrículos do Coração , Humanos , Silicones , Engenharia Tecidual/métodosRESUMO
BACKGROUND: Human engineered heart tissue (EHT) transplantation represents a potential regenerative strategy for patients with heart failure and has been successful in preclinical models. Clinical application requires upscaling, adaptation to good manufacturing practices, and determination of the effective dose. METHODS: Cardiomyocytes were differentiated from 3 different human induced pluripotent stem cell lines including one reprogrammed under good manufacturing practice conditions. Protocols for human induced pluripotent stem cell expansion, cardiomyocyte differentiation, and EHT generation were adapted to substances available in good manufacturing practice quality. EHT geometry was modified to generate patches suitable for transplantation in a small-animal model and perspectively humans. Repair efficacy was evaluated at 3 doses in a cryo-injury guinea pig model. Human-scale patches were epicardially transplanted onto healthy hearts in pigs to assess technical feasibility. RESULTS: We created mesh-structured tissue patches for transplantation in guinea pigs (1.5×2.5 cm, 9-15×106 cardiomyocytes) and pigs (5×7 cm, 450×106 cardiomyocytes). EHT patches coherently beat in culture and developed high force (mean 4.6 mN). Cardiomyocytes matured, aligned along the force lines, and demonstrated advanced sarcomeric structure and action potential characteristics closely resembling human ventricular tissue. EHT patches containing ≈4.5, 8.5, 12×106, or no cells were transplanted 7 days after cryo-injury (n=18-19 per group). EHT transplantation resulted in a dose-dependent remuscularization (graft size: 0%-12% of the scar). Only high-dose patches improved left ventricular function (+8% absolute, +24% relative increase). The grafts showed time-dependent cardiomyocyte proliferation. Although standard EHT patches did not withstand transplantation in pigs, the human-scale patch enabled successful patch transplantation. CONCLUSIONS: EHT patch transplantation resulted in a partial remuscularization of the injured heart and improved left ventricular function in a dose-dependent manner in a guinea pig injury model. Human-scale patches were successfully transplanted in pigs in a proof-of-principle study.
