Bioengineering of living heart valve substitute suitable for implantation into pediatric patients with heart valve diseases is an unsolved challenge in cardiovascular tissue engineering. In order to identify desirable material properties of such construct, the biomechanical, histological, histochemical and biochemical properties of fetal, perinatal and adult porcine heart valve leaflets have been systematically investigated. It have been shown that increasing in heart valve stiffness during ontogenesis correlates with accumulation of collagen type 1 and increasing level of collagen cross-linking. Tissue spheroids biofabricated from human fat tissue derived stem cells have been employed to engineer leaflet-like construct on compliant electrospun scaffold using self-assembly or tissue fusion approach. Incubation of tissue engineered heart valve leaflet construct with TGFβ as well as periostin transfection significantly increased stiffness of tissue engineered heart valve leaflet. These data demonstrated that the heart valve can be biofabricated by tissue fusion or self-assembly of closely placed tissue spheroids and that chemically and genetically induced accelerated tissue maturation and collagen deposition can enhance mechanical properties of tissue engineered leaflet. Further optimization of accelerated tissue maturation technologies could eventually lead to development of living autologous human heart valve tissue engineered construct with natural- like biomechanical properties suitable for pediatric cardiac patients.