Engineering a controlled cardiac multilineage co-differentiation process using statistical design of experiments

The heart is a complex organ composed of diverse cell types, whose interplay is crucial for development, tissue homeostasis, and disease progression. A potential approach to recapitulate this heterotypic multicellular environment is the co-differentiation of induced pluripotent stem cells (iPSCs) into multiple cardiac cell types, facilitating the unlocking of their full potential for regenerative medicine and drug development. However, the inherent complexity of co-differentiation, where multiple differentiation factors simultaneously influence the induction of multiple cell types, presents a significant challenge in achieving high controllability over the process toward the desired outcome. Thus, a robust strategy is essential for engineering a controlled co-differentiation process with broader applicability. Given the importance of heterotypic cellular proportions in facilitating proper interactions, we present a new strategy to engineer a controlled cardiac co-differentiation process from iPSCs using statistical design of experiments to simultaneously generate cardiomyocytes, mural cells, and endothelial cells, which are major constituents of the heart. We divided the process into two stages: progenitor cell induction and the subsequent trilineage co-differentiation, allowing for stage-specific optimization. Given that the performance of progenitor cell induction may critically influence the overall process performance, we carefully optimized activin A and CHIR-99021 using the sequential design of experiments to achieve approximately 95% induction efficiency of KDR+/PDGFR-α+ cardiogenic mesoderm cells from iPSCs with minimal batch-to-batch variability. In the trilineage co-differentiation stage, we developed unique multi-response models to delineate trilineage co-differentiation ratios within a defined parameter space of WNT signal inhibitor and vascular endothelial growth factor. This enabled the identification of potential conditions that steer co-differentiation toward desired cellular constitutions, a critical factor of effective cellular interplays. Repeated trilineage co-differentiation experiments confirmed the high process controllability, with a close match between actual and predicted differentiation ratios. Furthermore, cardiomyocytes from trilineage co-differentiation exhibited a more mature sarcomere gene expression profile than those from monolineage differentiation. These results highlight the effectiveness of our strategy for engineering a stem cell co-differentiation process and its applicability where multicellular interactions are crucial.