Assessment of the Optimal Generation Period and Size of Human iPSC-Derived Cardiac Organoids for Cardiotoxicity Drug Testing

Abstract

Abstract Background Human-induced pluripotent stem cell-derived cardiac organoids (hiPSC-COs) have emerged as valuable tools for cardiotoxicity drug screening, given their ability to recapitulate in vivo cardiac tissue functions and facilitate rapid assessment of drug stability to prevent toxicity. However, the lack of established criteria for the differentiation period and size during the generation of functional hiPSC-COs can introduce low accuracy in drug screening responses results. Hence, it is crucial to establish appropriate criteria for the generation period and size of hiPSC-COs to ensure reliable cardiotoxicity drug screening. Methods In this study, we generated different-sized hiPSC-COs in two types of microwell arrays through a one-stop generation method. The two-sized hiPSC-COs were continuously monitored until a stable cardiac beating rate was confirmed during the differentiation period. We evaluated and compared the functionality such as calcium transients at the selected differentiation day that showed a stable beating rate with a specific focus on determining the minimal differentiation period required for generating functional hiPSC-COs. A physiological test was conducted to verify the reactivity to the drug in hiPSC-COs according to the differentiation period and size. Subsequently, we conducted a cardiotoxicity drug screening test using compounds known to induce in vivo heart failure. Finally, characterization was analyzed by immunostaining assay to compare and confirm the phenotype of the two-sized hiPSC-COs at the selected differentiation period. Results During the differentiation period to generate hiPSC-COs, we identified the time point at which the smaller organoids among the two sizes of hiPSC-COs began to show a stable beating rate, which was an optimal period to lead to meaningful response results to cardiotoxicity drugs. Moreover, large organoids confirmed that cardiac properties disappeared as the differentiation period progressed, suggesting insight into the size limitation on the generation of hiPSC-COs for cardiotoxicity testing. Furthermore, an additional analysis method was proposed for subtle reactions that are difficult to confirm solely using the beating rate analysis in drug response testing. Conclusion We expect that these findings may contribute to the field of drug development by ensuring significant drug response results and enhancing the reliability of cardiotoxicity testing using hiPSC-COs.