PROJECT SUMMARY The immaturity of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) has emerged as a major challenge for their broad applicability. For this reason, extensive biological and engineering efforts are underway with the goal to generate mature, adult-like CMs from PSCs. However, owing to the lack of quantitative metrics to benchmark the maturation status of PSC-CMs, the efforts are being made largely on an ad hoc basis, and this leaves the degree and direction of PSC-CM maturation unclear. To address this, we have developed a quantitative metric based on an entropy concept that enables cross-comparison studies robust to experimental variability and species difference. With this finding, this proposal aims to apply entropy score to determine the status and trajectory of PSC-CM maturation achieved by molecular stimulation, tissue engineering, and in vivo transplantation, combined with functional and structural analysis. The use of entropy score is expected to reveal strengths and weaknesses of the ongoing approaches and inform us potential causes of maturation arrest and deviations. Thus, the proposed research will provide mechanistic insights into instructing PSC-CM to become adult CMs as well as quantitative insights into the progress made towards PSC-CM maturation in the field.