Bioinspired Manufacturing of hiPSC-based Therapy Products for application in Cardiovascular Regenerative Medicine

Background \u0026 Aim In vitro differentiation of human induced pluripotent stem cells into cardiomyocytes (hiPSC-CMs) is a crucial process to enable their application in cell therapy and drug discovery. Despite the remarkable efforts over the last decade towards the optimization of cardiac differentiation protocols, there are some technological challenges remaining, including the low scalability and differentiation yields. Additionally, generated hiPSC-CM are still immature, closely reminiscent of fetal/embryonic cells in what regards phenotype, structure and function. Our work aims to devise novel bioinspired strategies to improve the generation and functionality of hiPSC-CM. Noteworthy, we integrated structural and functional analyses of hPSC-CM with powerful “omics” technologies as to support bioprocess understanding and cell product characterization. Methods, Results \u0026 Conclusion We relied on the aggregation of hiPSC-derived cardiac progenitors to establish a scalable differentiation protocol capable of generating highly pure CM aggregate cultures. Whole-transcriptome and 13C-metabolic flux analyses demonstrated that a three-dimensional (3D) and agitated-based culture environment improves CM purity, functionality and metabolic maturation. We also assessed if alteration of culture medium composition to recapitulate in vivo substrate usage during heart development improved further hiPSC-CM maturation in vitro. By shifting hiPSC-CMs from glucose-containing to galactose- and fatty acid-containing medium, we promoted their fast maturation into adult-like CMs with higher oxidative metabolism, higher myofibril density and alignment as well as improved calcium handling and contractility. This study demonstrated for the first time that metabolic shifts during differentiation/maturation of hiPSC-CM are a cause, rather than a consequence, of the phenotypic and functional alterations observed. The bioinspired metabolic-based strategy established herein holds technical and economic advantages over the existing protocols due to its scalability, simplicity and ease of application. We are now applying the generated hiPSC-CM for the development of physiologically-relevant cardiac tissues. By modulating key environmental factors namely, electrical and physical stimuli, co-culture with other hiPSC-cardiac derivatives, biomaterials as well as components of the ECM, we were able to improve further hiPSC-CM maturation features and provide novel insight associated with heart regenerative mechanisms.