Origins and Proteomic Dynamics of Human Induced Pluripotent Stem Cell-Derived Extracellular Vesicles along the Temporal Trajectory of Cardiomyocyte Differentiation

Abstract Stem-cell-derived extracellular vesicles (EVs) that carry bioactive proteins and nucleic acids reflecting the parental cells offer a promising route for cell-free cardiac regenerative medicine. However, the origin, composition, and function of EVs derived from differentiating human induced pluripotent stem cells (iPSCs) during cardiomyocyte differentiation are largely unknown. We report a detailed, 4D label-free proteomic analysis of EVs at 17 consecutive time points and their parental cells at three crucial differentiation stages to investigate the origins and proteome dynamics of human iPSC-derived EVs along the temporal trajectory of cardiomyocyte differentiation. Our comprehensive proteomic profiling reveals dynamic oscillations in EV proteomes, reflecting alterations in protein expression in parental cells over the time course of the cardiomyocyte differentiation. Comparative proteomic analyses of EVs and their respective parental cells at three critical stages reveal that proteins associated with extracellular matrix organization and cell differentiation are preferentially sorted into EVs throughout differentiation, particularly at the postcardiac progenitor stage. However, proteins involved in gene expression and cellular metabolism are significantly retained in parental cells during differentiation, particularly at the precardiac progenitor stage. We investigated temporal proteomic changes between consecutive EVs, revealing regulatory proteomic dynamics during cardiomyocyte differentiation. Our study enhances our understanding of EV protein sorting during cardiomyocyte differentiation over time and the molecular mechanisms controlling each differentiation stage. It provides valuable insights for further studies exploring the functions of human iPSC-derived EVs isolated at different differentiation stages and their applications in advancing cell-free cardiac regeneration therapies.