Exploiting the human amniotic fluid stem cell secretome for cardiomyocyte renewal

Functional replacement of cardiomyocytes following injury represents a limiting aspect in cardiac regeneration. Given the extremely low intrinsic turnover rate of adult cardiomyocytes, their therapeutic renewal represents an unmet clinical need. Against such scenario, progenitor cell-based paracrine therapy has gained increasing attention in terms of candidate strategy to stimulate endogenous mechanism of repair and regeneration within the injured heart. Previous results from the group where I have carried out my PhD demonstrated that the secretome (namely the whole of the paracrine soluble factors that cells can release in vitro in their conditioned medium) of a specific subpopulation of human mesenchymal stromal cells from the amniotic fluid (hAFS: human amniotic fluid-derived stem cells) can exert significant cardioprotective effects and stimulate resident cardiomyocytes to engage into the DNA duplication step of the cell cycle in a preclinical adult mouse model of acute myocardial infarction. While such results advocate the hAFS relevant paracrine potential, further detailed studies have been necessary to better characterize the cardio-active capacity of the hAFS secretome. This has been pursued by (i) profiling the hAFS secretome formulations, (including the particulate fraction of their secreted extracellular vesicles - EVs) more in details, and (ii) by means of a more responsive experimental model represented by the neonatal mouse model of myocardial infarction. In this PhD project attention has been driven towards the extensive investigation of the cardiac regenerative paracrine effects exerted by two different sources of human progenitor cells: II trimester of gestation fetal hAFS, obtained from leftover samples of amniocentesis prenatal screening and term III trimester perinatal hAFS, isolated from clinical waste amniotic fluid from scheduled caesarean delivery. The hAFS secretome has been considered as either the total cell-conditioned medium (hAFS-CM), as compared to the extracellular vesicles sub-fraction within it (hAFS-EVs). Gestational stage, underlying the comparison of II trimester fetal hAFS versus III trimester perinatal hAFS, did not influence either the cell phenotype or the yield of the secretome formulations. While fetal and perinatal hAFS-EVs presented similar size distribution with a stable miRNA core in their cargo, proteomic and cytokine/chemokine profiling of the hAFS-CM highlighted some discrepancies between the formulations. Fetal hAFS-EVs resulted the secretome fraction endowed with the most stimulatory paracrine potential in sustaining cardiomyocyte cell cycle progression from S- up to M-phase with evidence of cytokinesis events. This was further supported by decreased expression of Cofilin-2 a pivotal player in cardiomyocyte cytoskeleton re-arrangement as required for cell-cycle re-entry. Fetal hAFS-EVs resulted particularly enriched in Agrin -a well-known mediator of cardiac regeneration in the neonatal mouse heart- as putative functional role mediator of the paracrine cardiogenic effects observed in vitro. Similar findings were obtained in vivo in a 4-days-old mouse model of myocardial infarction where fetal hAFS-EVs supported to some extent resident cardiomyocyte S-M phase transition in the remote myocardium within 3 days from injury and acute treatment. These encouraging results suggest that EVs obtained by immature fetal hAFS may retain promising cardio-active paracrine potential for myocardial renewal. Nonetheless, additional analyses are required to pinpoint the detailed molecular mechanism(s) of function in order to optimize such effect.