TRANSDIFFERENZIAMENTO DI CELLULE STAMINALI MESENCHIMALI DI RODITORE IN CELLULE DEL SISTEMA NERVOSO CENTRALE

One of the most important challenges in modern medicine is the identification of cell therapy protocols, enabling identification of personalized treatment strategies. WIthin this context, adult stem cells (ASC) have a large applicative potential. Contrary to ESCs (embryonic stem cells) and IPSCs (induced pluripotent stem cells), ASCs can be easily extracted from different tissues (bone marrow, skin, adipose tissue, muscle) without raising ethical issues . Moreover, they can be used for autologous transplantation, eliminating the complications associated with autoimmune reactions. Mesenchymal stem cells (MSC,), are an ASC population present in the bone marrow, adipose tissue and other tissues. MSCs are readily available and are able to self-replicate and differentiate, supported by the presence of appropriate stimuli, into cell lines derived from mesodermal lineage (osteoblasts, chondrocytes, adipocytes, and muscle cells). In the last decade it has also been observed that MSCs are able to trans-differentiate into cell types of ectodermal and endodermal origin (transdifferentiation).. The purpose of my work was to develop and define the conditions which can induce trans-differentiation of MSCs extracted from rat adipose tissue toward the neuronal phenotype. The study is part of a major project of regenerative medicine focused on identifying new strategies aimed at restoring functionality in degenerated brain tissue. Initially, MSCs have been characterized by the induction of osteoblastic differentiation, one of their physiological differentiations, and their interaction with biocompatible materials ( titanium dioxide) was analyzed, in order to assess their possible application in medicine for the creation, for example, of prothesic solutions. Subsequently, we sought to devise a GMP compliant cell culturing medium for MSC proliferation, crucial in order to obtain a clinically relevant cell number to differentiate following isolation from adipose tissue. For that reason we tried to induce the differentiation of MSCs into a neural phenotype; a first approach has been to apply protocols already known in literature for the differentiation of different types of stem cells toward the neuronal phenotype.. We considered both the direct differentiation protocols, as well as those that encompassed intermediate stages (sphere-forming). The protocols of differentiation by sphere formation resulted in differentiated MSC expressing glial markers (GFAP), but the protocol was too long(30 days) and a number of differentiated cells very low.. A second approach has been to develop a proprietary differentiation medium (NZ4) using the knowledge and expertise gained from the literature and from the negative results previously obtained The cells maintained in NZ4 differentiation medium , showed a clear morphological change and a high vitality; moreover they expressed both markers of specific neurons in the early stages of development (nestin, doublecortin) as well as markers expressed by mature neurons (βIIItubulin, VAMP2, Sinaptotagmin). Furthermore, the efficiency of the differentiation protocol has been functionally characterized by means of qualitative analysis of the dynamics of intracellular calcium and with quantitative analysis of the resting membrane potential The functional characterization clearly showed that MSC exposed to NZ4 differentiation medium have comparable behavior as primary neurons undergoing in vitro maturation. Given the future direction of the project will be to inject in an animal model the cell undergoing differentiation in order to attempt a partial recovery of the damaged neuronal tissue (i.e. ischemia) we characterized the behavior of MSC encapsulated in a biocompatible bioresorbable hydrogel matrix. The cells were encapsulated for different time periods, recovered, and heir survival as well as maintenance of stemness potential following retrieval was assessed . There are still other issues to be clarified n in particular, it will be necessary to verify in an in vivo setting the the efficacv of the differentiation protocol as well as to characterize the process of MSCs homing and to perform more detailed studies on behavioral changes of the same animal model. However, preliminary data obtained during this thesis allows to deepen the knowledge concerning the differentiation process of MSC from adipose tissue toward a neuronal phenotype.