Impact of secretome containing extracellular vesicles from human induced pluripotent stem cell-derived cardiomyocytes

Cell transplantation therapy using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has shown promise for ischemic heart disease. Although paracrine effects mediated by the secretome are recognized as key mechanisms underlying the therapeutic effects of hiPSC-CMs, dynamic changes in the secretome profile during the manufacturing of hiPSC-CMs remain poorly understood. This study was aimed at elucidating the secretome dynamics of clinical-grade hiPSC-CMs to identify their "mode of action" (MoA) and "candidate quality attributes."

We integrated multiomics profiling, including total RNA-sequencing, proteome analysis, and extracellular vesicle (EV)-derived microRNA-sequencing, with functional assays (cell proliferation, cell migration, and endothelial tube formation) to analyze samples obtained at different time points in the hiPSC-CM manufacturing process (Day 4-25).

High-purity hiPSC-CMs (>95% cardiac troponin T-positivity) exhibited distinct transcriptomic maturation between Day 16 and Day 25, characterized by the upregulation of cardiac marker genes. Proteomic clustering revealed four distinct stages, with functional transition from proliferation-centric signaling to tissue-repair signaling. Notably, the purified Day 25 secretome showed a qualitative shift toward a platelet-derived growth factor- and stromal cell-derived factor 1-rich profile. Simultaneously, EVs from the Day 25 secretome were enriched in "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 the formation of mature, highly branched endothelial tubes compared with the secretome from earlier stages.

The hiPSC-CM secretome containing EVs undergoes programmed evolution during manufacturing, with functional transition from promotion of undifferentiated growth to 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 candidate quality attributes to ensure the potency and consistency of clinical-grade cardiac products.