The Human Amniotic Fluid Stem Cell Secretome as a powerful tool to unlock endogenous mechanism of cardiac repair and regeneration

Human amniotic fluid stem cells (hAFS) are immature fetal mesenchymal progenitors with promising paracrine potential, as previously shown by a study in a preclinical rat model of ischemia/reperfusion injury. Nevertheless, little is known about the comprehensive analysis of their secretome, which is represented by the whole of soluble secreted factors and extracellular vesicles (EV) released in the cell conditioned medium (hAFS-CM). The aim of this PhD project is to characterise in details the hAFS secretome paracrine modulatory capacity, including the role of their extracellular vesicles, for future cardiac regenerative medicine. The cardioprotecive potential of the hAFS-conditioned media (hAFS-CM) has been first assessed in a model of doxorubicin-induced cardiotoxicity in vitro. Indeed, the anthracycline doxorubicin is widely used in oncology, but it may cause cardiomyopathy with dismal prognosis that cannot be effectively prevented. Following hypoxic preconditioning, hAFS-CM demonstrated to actively antagonizes senescence and apoptosis of cardiomyocytes and cardiac progenitor cells, two major features of doxorubicin cardiotoxicity. In order to fully understand the pro-active role of hAFS-EV in mediating paracrine regenerative effects, the second part of my PhD project has been dedicated to analyse the hAFS-derived EV (hAFS-EV) potential in providing proliferative, pro-survival, immunomodulatory and angiogenic effects. In particular, the role of hypoxic preconditioning on hAFS cells in priming their EV has been here evaluated as well. In vitro analyses on target cells defined their role as biologically mediators of paracrine effects by direct transfer of regenerative microRNAs, while the in vivo analysis in a preclinical mouse model of skeletal muscle atrophy study confirmed their modulatory role in decreasing pathological inflammation. Finally, I confirmed the cardioactive profile of the hAFS secretome in a preclinical mouse model of myocardial infarction (MI), by specifically focusing on the enhancement of cardiac repair and the reactivation of the endogenous mechanism of regeneration via stimulation of cardiac progenitor cell in situ and of resident cardiomyocyte proliferation. Intra-myocardial injection of hAFS-CM soon after MI provided substantial cardioprotection, curbed down inflammation in the short term, while also sustaining angiogenesis and boosting cardiomyocyte proliferation. Notably, the hAFS secretome also induced the increase of endogenous epicardial progenitor cells within a week from MI, with a possible more specific role of hAFS-EV in unlocking their activation as well as rescuing cardiac function. These encouraging findings suggest the hAFS secretome as an appealing source of therapeutic paracrine factors for the development of a future medicinal advanced product in cardiac regenerative medicine, so to provide cardioprotection following drug-derived injury or an ischemic insult, and improve cardiac repair while restoring the endogenous regenerative program within the heart.