Isolamento e caratterizzazione di sottopopolazioni ES-simili dal sangue di cordone ombelicale e periferico umani e dal midollo osseo murino

During the last few years much progress has been made in the knowledge of adult and embryonic stem cell biological properties. The ability to isolate and culture in conditions that allow the expansion of these cells without their losing stem potential, has helped in using stem cells as a tool to treat patients suffering from certain cellular degenerative diseases or affected with a specific cellular population loss of functionality. Because of their wide potentialities, embryonic stem cells (ES) obtained from blastocyst inner cell mass, appear to have the best chance as a tool to treat degenerative diseases affecting tissues of different embryonic origin. Biological difficulties (such as the risk of uncontrolled proliferation, graft versus host disease, the need to set culture conditions free of non-human compounds) and, above all, legal and ethical problems, seem to prevent the therapeutic application of such cells. The identification and characterization of an ES-like cell population in adult tissues might be of great help to exploit these cells in the medical field. In this thesis I show that a cellular population featuring certain molecular ES cell properties, may be isolated from human cord blood after depletion of all differentiated cells but monocytes, followed by the positive selection of cells bearing p75 neurotrophin receptor (p75NTR). Cytofluorimetric assays revealed that a fraction of p75NTR+ cells express SSEA3 and SSEA4, two typical human ES cell glycolipidic markers. Real Time RT-PCR assays showed that, following the selection for p75NTR+, a cell population is obtained that expresses very high levels of Oct4 and Nanog, two transcription factors whose importance in ES stemness and self-renewal is largely documented. Further increase of Oct4 and Nanog expression level is obtained following depletion of cells bearing Mac-1, a typical monocytes marker. A similar study has been made in human peripheral blood and mouse bone marrow. Data obtained by Real Time RT-PCR assays, have shown that in two out of four samples of adult peripheral blood analysed by the above selection method, following isolation of p75NTR+ cells, a cell population expressing high levels of Oct4 and Nanog was obtained. In 4-5 weeks-old mouse bone marrow, the highest expression of Oct4 and Nanog has been detected in p75NTR+/Mac-1- cells, while, in older mice (13-14 weeks), such expression increase has been detected in p75NTR+/Mac-1+ cells. Further studies are needed to evaluate whether these cells, in addition to bearing ES cell molecular markers, also have functional ES-like properties and can be classified as true ES-like cells