Unravelling the ageing-reversal potency of stem cell-derived extracellular vesicles in a rat model of premature cardiac senescence

Cardiosphere-derived cells (CDCs) and the extracellular vesicles they release (CDC-EVs) have demonstrated to induce rejuvenation and to improve cardiac structure and function, but their efficacy may vary among donors and there is still a lack of adequate potency assays. We aimed to identify parameters that could easily predict the ageing-reversal potency of CDC-EVs. CDCs derived from cardiac tissue of 34 human donors (age range: 3-months to 81-years old) were characterized in terms of their phenotypical and biological properties. The anti-senescent activity of the CDC-EVs was assessed in vitro using a predesigned matrix assay with several ageing-related markers. We found that while CDC-donors chronological age was not determinant, the degree of CDC senescence in culture showed a strong correlation to most CD[C]s bioactive properties. However, CDC senescence alone was insufficient to predict CDC-EV in vitro potency. Thus, we scored the potency of the CDC-EVs based on their performance in the newly designed in vitro matrix assay and classified them as more-(P-EVs) and less-potent (NP-EVs). We then tested P- and NP-EVs in a rat model of premature cardiac ageing and found that only the P-EVs reduced senescence-associated GLB1 gene expression in the heart and tended to protect it from hypertrophy development and fibrosis. On the contrary, NP-EVs failed to induce any improvements and negatively affected cardiac hypertrophy, fibrosis, and perfusion. In conclusion, despite CDC-EV anti-ageing potency cannot be predicted by the chronological age of the donors or CDC senescence as surrogate markers, we propose an in vitro potency assay that could be used to evaluate allogenic EV suitability before its use in the treatment of cardiac ageing.