Assistant Professor Nagoya University Nagoya, Aichi, Japan
Abstract: Pluripotent stem cell-derived cardiomyocytes hold great potential for regenerative medicine and drug screening, but their immaturity remains a major barrier to practical applications. During cardiac development, cardiomyocytes are induced together with non-cardiomyocyte cells from cardiac mesoderm cells (CMCs), and their interactions play a crucial role in functional maturation. Inspired by this principle, we aimed to engineer a co-differentiation process for the simultaneous generation of three major cardiac cell types–cardiomyocytes, mural cells, and endothelial cells–from induced pluripotent stem cells (iPSCs). The key challenge was whether these cell types could be co-differentiated in a controlled manner to achieve a desired cell composition favorable for cardiomyocytes. To address this, we employed design of experiments (DoE), a powerful statistical process engineering approach. Our study consisted of developing two subprocesses: (1) differentiation of iPSCs into CMCs and (2) differentiation of CMCs into the three cardiac cell types. For (1), we used response surface methodology (RSM) with CHIR99021 and Activin A, achieving approximately 95% efficiency of CMCs. For (2), we applied RSM with Wnt inhibitors and VEGF to construct predictive models for co-differentiation into the three cell types, enabling us to select ideal co-differentiation conditions. Validation experiments confirmed that the predicted and actual outcomes were comparable, underscoring process controllability. Furthermore, gene expression analysis on day 40 of differentiation revealed enhanced maturation for cardiomyocytes from the controlled co-differentiation compared to those without non-cardiomyocyte cells. The results highlight the effectiveness of our approach in engineering controlled cardiac co-differentiation processes and suggest broader applicability for developing other differentiation processes where heterotypic cell interactions are important.
