Advancing Human iPSC-Derived Cardiomyocyte Hypoxia Resistance for Cardiac Regenerative Therapies through a Systematic Assessment of In Vitro Conditioning

Acute myocardial infarction (MI) is a sudden, severe cardiac ischemic event that results in the death of up to one billion cardiomyocytes (CMs) and subsequent decrease in cardiac function. Engineered cardiac tissues (ECTs) are a promising approach to deliver the necessary mass of CMs to remuscularize the heart. However, the hypoxic environment of the heart post-MI presents a critical challenge for CM engraftment. Here, we present a high-throughput, systematic study targeting several physiological features of human induced pluripotent stem cell-derived CMs (hiPSC-CMs), including metabolism, Wnt signaling, substrate, heat shock, apoptosis, and mitochondrial stabilization, to assess their efficacy in promoting ischemia resistance in hiPSC-CMs. The results of 2D experiments identify hypoxia preconditioning (HPC) and metabolic conditioning as having a significant influence on hiPSC-CM function in normoxia and hypoxia. Within 3D engineered cardiac tissues (ECTs), metabolic conditioning with maturation media (MM), featuring high fatty acid and calcium concentration, results in a 1.5-fold increase in active stress generation as compared to RPMI/B27 control ECTs in normoxic conditions. Yet, this functional improvement is lost after hypoxia treatment. Interestingly, HPC can partially rescue the function of MM-treated ECTs after hypoxia. Our systematic and iterative approach provides a strong foundation for assessing and leveraging in vitro culture conditions to enhance the hypoxia resistance, and thus the successful clinical translation, of hiPSC-CMs in cardiac regenerative therapies.