Dottorato in MEDICINA SPERIMENTALE

Titolo della tesi: Adipose-derived stem cells (ASCs) in regenerative medicine: epigenetic and molecular approaches to optimize the therapeutic potential mediated by cells and secretome

Adipose-derived mesenchymal stem cells (ASCs) are a multipotent stem cell population that resides in the SVF of adipose tissue. They represent a valid therapeutic option for clinical application in several diseases, due to their ability to repair damaged tissues and to mitigate the inflammatory/immune response. Considering the role of epigenetic mechanisms on the regulation of gene expression and differentiation potential, we investigated the effects of the DNA methyltransferase inhibitor 5-Azacytidine (5-aza) on several biological processes of ASCs, such as proliferation, migration, adipogenic differentiation and senescence. Indeed, the data obtained demonstrated that 5-aza determines a reduction in both clonogenic and migration potential of ASCs, as well as an increase of their differentiation towards the adipogenic lineage. At the same time though, the pre-treatment induced cellular senescence, denoting the delicate balance in place in the regulation of the diverse biological functions of stem cells . Moreover, ASCs exert their therapeutic action especially through their paracrine activity, by secreting soluble factors and extra-vesicles collectively known as secretome. Hence, we focused on ASCs secretome as an alternative therapeutic tool to cell therapies. Indeed, boosting ASCs features prior to administration represents an attractive strategy to overcome the limited efficacy of naïve ASCs. By modifying the composition of the secretome we are also able to modulate the therapeutic effects of ASCs on target cells. Transfection with microRNAs, such as miR-125b which is known for regulating both proliferation and differentiation, in addition to its effects on inflammation and immune response, represents a feasible strategy to potentiate ASCs secretome, leading to a modulation of its cytokines composition, as observed through protein arrays. In view of a secretome-based therapy enhanced with microRNAs, we then focused on Neuropilin 1 (NRP1) as a receptor for miRNAs, confirming its importance in the binding and entry of extracellular miR-125b through silencing experiments. Finally, through co-cultures of PBMCs with miR-125b-enriched ASC-conditioned medium, we evaluated the ability of the boosted secretome to induce a modulation of PBMCs activation, observing an impairment of T cells induced by miR-125-enriched secretome, this confirming our hypothesis of an immunomodulatory action mediated by this potentiated secretome. In conclusion, the data obtained help to shed light on the potential risks and advantages of in vitro epigenetic and molecular approaches to improve existing therapeutic strategies involving the clinical use of ASCs and, at the same time, to develop new ones by boosting the secretome produced by these cells, thus combining the powerful immunomodulatory capacities of ASCs with the advantages of a cell-free therapy.