Biochemical and mechanical signals enabling cardiac regeneration can be elucidated by using in vitro tissue engineering models. We hypothesized that human insulin-like growth factor-I (IGF-I) and 3-dimensional (3D) dynamic microenvironment could enhance the survival and differentiation of adipose tissue-derived stem cells (ADSCs). In this study, ADSCs were cultured on 3D porous scaffolds with or without plasmid DNA PIRES2-IGF-I in cardiac media, in static culture dishes, and in a spinning flask bioreactor, respectively. Cell viability, formation of cardiac-like structure, expression of functional proteins, and gene expressions were tested in the cultured constructs on day 14. The results showed that dynamic microenvironment enhanced the release of plasmid DNA; the ADSCs can be transfected by the released plasmid DNA PIRES2-IGF-I in scaffold. IGF-I showed beneficial effects on cellular viability and increase of total protein and also increased the expressions of cardiac-specific proteins and genes in the grafts. It was also demonstrated that dynamic stirring environment could promote the proliferation of ADSCs. Therefore, IGF-I, expressed by ADSCs transfected by DNA PIRES2-IGF-I incorporated into scaffold, and hydrodynamic microenvironment can independently and interactively increase cellular viability and interactively increase the expression of cardiac-specific proteins and genes in the grafts. The results would be useful for developing tissue-engineered grafts for myocardial repair.