THE USE OF TEN ELEVEN TRANSLOCATION (TET) ENZYMES TO ERASE SIGNS OF AGING.

The world is aging rapidly, and, in the EU, the share of population aged over 60 will reach 30% by 2050. As people live longer, frailty increases and the risk of developing age-related diseases goes up. Among the several options available, regenerative medicine provides a valid strategy for the prevention and reduction of ageing and great promises may be identified in the pathways activated along cell epigenetic erasing as well as humoral signaling distinctive of the cell microenvironment. In this thesis, I investigate whether ageing may be counteracted using two different approaches: • A) I explored the possibility to rejuvenate cells using TET downstream miRNAs, miR-200a, miR-200b, miR-200c, miR-145 and miR-429, that are known to be actors in epigenetic erasing and that belong to the miR-200 family • B) I evaluated whether the secretome obtained from young cells may be rich in soluble factors that are able to enforce a juvenile phenotype and endowed with an anti-senescent action. I adopted an experimental design based on the use of fibroblasts obtained from healthy individuals aged 35 and 96 years, from patients affected by the Hutchinson Gilford Progeria Syndrome (HGPS) and on fibroblasts that were artificially aged via exposure to UV and hydrogen peroxide, and arbitrarily defined senescence induced (SI) All these cell groups were scored for well characterized ageing specific molecules that were used as markers to monitor the rejuvenating effects of the approaches described above. The data obtained demonstrated that the treatment with the cocktail of miR-200b and miR-200c have an anti-ageing effect with the acquisition of a self-renewing state and the reduction of all the ageing markers analyzed. A similar rejuvenating effect was obtained with the use of secretomes isolated from young cell cultures. Among the several soluble factors that may be responsible for this effect, I hypothesized the possible involvement of exosomes. In agreement with this, when the secretome was subjected to an exosome depriving chromatographic separation, the rejuvenating effect was significantly decreased. In a parallel, although strictly related series of experiments, I set out to create an artificial implantation system, involving both the trophoblast compartment as well as the endometrium, where I could study in a highly controlled in vitro environment the decline in reproductive performance connected to the steadily increasing maternal age and women empowerment. To this purpose I generated trophectoderm-like lineages from young, aged and HGPS fibroblasts that were epigenetically converted to cytotrophoblast and syncytiotrophoblast cells. All cell groups showed positivity for specific trophectoderm markers such as CDX2, PAPPA, PSG6 and GCM1, but senescent cells displayed a lower differentiation efficiency and decreased expression rate. Unfortunately, due to the pandemics, I was not able to conclude the experiments for this model, due to the impossibility to collect endometrial samples from the local hospital. In conclusion, the experiments carried out during my PhD program have allowed to demonstrate the possibility to counteract the ageing process in vitro in different cell types. Interestingly, rejuvenation was achieved both using TET downstream miRNAs and soluble factors present in young cell secretomes, suggesting the possibility to harness ageing via the activation of specific molecular pathways as well as with the exposure to the appropriate cell microenvironment.