SVILUPPO DI TECNICHE PER L'ISOLAMENTO, LA CARATTERIZZAZIONE E IL DIFFERENZIAMENTO DI CELLULE STAMINALI MESENCHIMALI PER UN USO IN MEDICINA RIGENERATIVA

My PhD project aims at developing innovative technologies allowing a complete and perfect recovery of the tissue from both a physiological and a mechanical point of view by taking advantage of the regenerative properties of the stem cells, focusing especially on articulations and bones damages. The stem cells are a cell population physiologically involved in the processes of tissue repair and regeneration therefore would suit well with a physiological, patient-specific regeneration of a damaged tissue. In our project, we will focus on the mesenchymal stem cells (MSCs) as they can differentiate into different cell lineages, and in particular into osteocytes and chondrocytes. The implantation of differentiated cells obtained in vitro from MSCs isolated from biopsies taken from the patient would prevent any problem of graft rejection. The hMSCs can be found in the bone marrow and the adipose tissue. In this case, they are called Adipose-like stem cells (hASCs), and share common characteristics with the hMSCs as it has already been described in the literature and in my project too: they have the same immunophenotypical profile (Glycophorine, CD31, CD34 and CD45 negative, CD73, CD90, CD105, CD117, CD271, and Stro-1 positive), and can give rise to the same cell lineages, such as osteocytes, chondrocytes and adipocytes cells. The different methods to isolate the hMSCs and the hASCs from human biopsies have been valuated and improved. hMSCs have been isolated from bone marrow samples, separated from the other cells on a Ficoll gradient and enriched with a cd271+ column, a specific marker of hMSCs before the final selection made by adherence. The hASCs have been isolated from lipo aspirate and after some protease treatments simply selected by adherence. Compare to bone marrow aspirate, the lipo-aspirate is a less invasive technique and gives a better efficiency of SCs isolation, the material to start with being more important. The protocol to isolate hASCs is as well faster and cheaper compare to the one used for the hMSCs. And as both hMSCs and hASCs in our study had the same pattern of expression of the staminality markers, we thought the hASCs were the most appropriate to use in our study. The experiments of osteocytes and chondrocytes differentiation using hMSCs and hASCs isolated from patients, suggest that the hASCs are most efficient to differentiate in osteocytes, as the markers of differentiation seem to be expressed earlier in the process. In some preliminary experiments, the hASCs have been seeded on an osteochondral biomimetic scaffold (Fin-Ceramica Faenza SpA) and we could see the presence of the cells already colonising the scaffold after 4h, before being induced for osteogenic differentiation for 12 days. The strong osteopontin signal visible suggests that the differentiation process already started and was almost completed, earlier than what we observed in vitro without the scaffold. This will be repeated leaving the cells to differentiate longer, and try as well to differentiate the ASCs in chondrocytes, still using the scaffold. In the future, different in vivo differentiation experiments where the scaffold will be grafted in mice will be performed. To evaluate the expression level of several genes involved in the different phases of the osteogenic differentiation process, we performed a day by day gene profiling analisis by Real-Time qPCR. This detailed study informed us on the regulation of the genes during the different phases, early, intermediate and late, of the osteogenic process. It gave us a more complete vision compare to what you can find in the literature.