Esposito, Maria Teresa (2009) In vitro and in vivo characterization of murine bone marrow stromal stem cells: self renewal, differentiation, tumorigenic potential. [Tesi di dottorato] (Unpublished)

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The use of adult stem cells for applications such as tissue engineering and gene therapy is one of the major challenge approaches of regenerative medicine. In the field of adult stem cells, bone marrow stromal cells (BMSCs) are very promising candidates. The ability to move forward the use of these cells in therapy depends on the setting up of mammalian models, necessary to evaluate pre-clinical efficacy and standardize procedures. Precisely, murine BMSCs (mBMSCs) characterization is a major breakthrough that can benefit from numerous established genetic models of human diseases now available in this organism. In particular, C57Bl/6 strain is frequently used to prepare transgenic mice because it produces a large number of high-quality embryos and super-ovulate in response to hormone injections. However, the BMSC is a cell type better studied and characterized in humans than in mouse and, although in the last few years numerous protocols have been developed to isolate these cells, some problems still remain. In the present study, I define, for the first time, conditions for optimizing the yields of cultures enriched for specific progenitors of bone marrow. I isolated four different sub-populations of mBMSCs using four distinct culture conditions, supernatants from culture of bone fragments, marrow stroma cell line MS-5, embryonic fibroblast cell line NIH3T3 and a cocktail of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). These cells show very interesting features in immunophenotype, self-renewal ability and differentiation potency. In particular, using NIH3T3 conditioned medium, we obtained cells that show impairment in osteogenic and chondrogenic differentiation, whereas retaining a very high adipogenic potential during passages. These results indicate that the choice of the medium used for isolation and expansion of mBMSCs is extremely relevant in order to enrich the culture of desired specific progenitors. A common characteristic among these sub-populations of mBMSCs was the expression of a well known marker of undifferentiated embryonic stem cells, namely Nanog. Recent studies have demonstrated the expression of Nanog in BMSCs and its effect on proliferation and osteogenic and chondrogenic abilities, however its role in these cells is still unknown. During a six- month period as visitor in the laboratory coordinated by Dr Nicole Horwood, at Imperial College (London, UK), I have generated vectors expressing Nanog sense and antisense in order to analyze the effect of over-expression and down-regulation of Nanog on migration, senescence, apoptosis and tumorigenesis in BMSCs. Preliminary data suggest that over-expression of Nanog does not seem to efficiently reduce the number of senescent cells; however, Nanog over-expressing cells showed a decrease in the expression of p53, a well known tumor-suppressor gene and acquired the ability to grow in soft agar, an in vitro hallmark of tumorigenic cells. On the other hand, down-regulation of Nanog in BMSCs enhances migration ability, and induces an increase in the expression of CCL5 (Rantes) and CXCL10 (IP10, interferon γ inducible protein-10), which have been reported to enhance motility, invasion and metastasis of breast cancer cells and to have a potent thymus-dependent antitumoral effect. This work suggest that defined levels of Nanog could affect BMSCs properties and indicate a further need of a better understanding of the biology of these cells which are already being used in several clinical trials.

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