Secretome analysis during human induced pluripotent stem cell-derived cardiomyocyte differentiation

Introduction: Cell transplantation therapy using human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) is a promising treatment for ischemic heart disease. While paracrine effects mediated by the secretome are recognized as a key therapeutic mechanism, the dynamic changes in the secretome profile during the manufacturing process remain poorly understood. This study aimed to comprehensively elucidate the secretome dynamics of clinical-grade hiPSC-CM to identify their "Mode of Action" (MoA) and candidate quality attributes (CQAs). Methods: We integrated multi-omics profiling-including total RNA-seq, proteome analysis, and extracellular vesicle (EV)-derived miRNA-seq-with functional assays (proliferation, cell migration, and tube formation) using samples obtained across time points of the hiPSC-CM manufacturing process (Day 4-25). Results: High-purity hiPSC-CM (>95% cTnT positive) exhibited a distinct transcriptomic maturation between Day 16 and Day 25, characterized by the upregulation of cardiac maker genes. Proteomic clustering revealed four distinct stages, showing a functional transition from proliferation-centric signaling to tissue-repair signaling. Notably, the purified Day 25 secretome showed a qualitative shift toward a PDGF and SDF-1 rich profile. Simultaneously, EVs from Day 25 were enriched with "myomiRs" (miR-133b, -208b, -499b) and multiple anti-proliferative miRNAs (let-7e-5p, miR-145-5p). Functionally, the Day 25 secretome significantly enhanced mesenchymal stem/stromal cell (MSC) migration and promoted mature, highly branched endothelial tube formation compared to earlier stages. Conclusions: The hiPSC-CM secretome undergoes a programmed evolution during manufacturing, shifting from promoting undifferentiated growth to orchestrating complex tissue repair through stable angiogenesis and MSC recruitment. These findings establish a molecular foundation for the MoA of hiPSC-CM therapy and provide critical CQAs for ensuring the potency and consistency of clinical-grade cardiac products.