The fabrication of biomimetic heart muscle suitable for pharmaceutical compound evaluation and disease modeling is hindered by limitations in our understanding of how to guide and assess the maturity of engineered myocardium in vitro. We hypothesized that tissue architecture serves as an important cue for directing the maturation of engineered heart tissues and that reliable assessment of maturity could be performed using a multi-parametric rubric utilizing cardiomyocytes of known developmental state as a basis for comparison. Physical micro-environmental cues are recognized to play a fundamental role in normal heart development, therefore we used micro-patterned extracellular matrix to direct isolated cardiac myocytes to self-assemble into anisotropic sheets reminiscent of the architecture observed in the laminar musculature of the heart. Comparison of global sarcomere alignment, gene expression, and contractile stress in engineered anisotropic myocardium to isotropic monolayers, as well as, adult ventricular tissue revealed that anisotropic engineered myocardium more closely matched the characteristics of adult ventricular tissue, than isotropic cultures of randomly organized cardiomyocytes. These findings support the notion that tissue architecture is an important cue for building mature engineered myocardium. Next, we sought to develop a quality assessment strategy that utilizes a core set of 64 experimental measurements representative of 4 major categories (i.e. gene expression, myofibril structure, electrical activity, and contractility) to provide a numeric score of how closely stem cell-derived cardiac myocytes match the physiological characteristics of mature, post-natal cardiomyocytes. The efficacy of this rubric was assessed by comparing anisotropic engineered tissues fabricated from commercially-available murine ES- (mES) and iPS- (miPS) derived myocytes against neonatal mouse ventricular myocytes. The quality index scores calculated for these cells revealed that the miPS-derived myocytes more closely resembled the neonate ventricular myocytes than the mES-derived myocytes. Taken together, the results of these studies provide valuable insight into the fabrication and validation of engineered myocardium that faithfully recapitulate the characteristics of mature ventricular myocardium found in vivo. These engineered tissue design and quality validation strategies may prove useful in developing heart muscle analogs from human stem cell-derived myocytes that more accurately predict patient response than currently used animal models. / Engineering and Applied Sciences