Comparative study of novel maturation pathway in iPSC- - derived cardiomyocytes via FOXO1/FOXM1 reveals improved maturation strategies

Abstract

Ischemic heart disease affects a significant part of the world’s population. Induced pluripotent stem cell-cardiomyocyte (iPSC-CM) transplantation strategies are attractive treatments for ischemic heart disease due to their repair of endogenous cardiac function. However, cardiac remuscularization strategies are still under development due to side effects that lead to life-threatening arrhythmias associated with an immature iPSC-CM phenotype. Here, we improved iPSC-CM maturation in vitro by mimicking postnatal signaling switches to overcome arrhythmias after iPSC-CM transplantation. We compared different soluble factor-based treatments in 3D cell culture to improve current iPSC-CM maturation protocols by characterizing key parameters of CM maturation. Such parameters included mRNA and protein expression of sarcomere and ion channel genes measured via qPCR and flow cytometry, oxygen consumption rate via Seahorse XF cell mito stress test, and contractility via multielectrode array. We showed iPSC-CM in prolonged 3D cultures are still responsive to FOXO1/FOXM1 alterations, suggesting a new way to improve iPSC-CM maturation in vitro in combination with 3D and prolonged culture. Our ion channel expression and multielectrode array data showed relevant iPSC-CM maturation after P53 activation and FOXM1 inhibition when compared to other treatments. We also found both P53 activation and FOXO1 activation are comparable in inducing metabolic maturation in iPSC-CM. Lastly, combining P53 activation with other maturation methods, like mTOR inhibition, offered improved maturation in 3D, representing a new iPSC-CM maturation strategy. Thus, we concluded FOXO1 activation/FOXM1 inhibition is a novel iPSC-CM maturation pathway, although the P53 pathway is more beneficial to iPSC-CM maturation in comparison to other pathways.