Abstract
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Abstract
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Kit receptor in the regulation of MSCs: protein expression
during adipogenic differentiation.
Mesenchymal Stem Cells or Marrow Stromal Cells (MSCs) are multipotent stromal cells that can differentiate, both in vitro and in vivo, along different cell lineages, including the osteogenic, condrogenic, adipogenic and myogenic ones. They have migratory ability and produce a wide spectrum of trophic and/or immunoregulatory molecules. Their major source is the bone marrow, where they represent the 0.01- 0.001% of the nucleated cells, however they reside also in most tissues. For these properties and the possibility to easily isolated and expanded them ex vivo, they attracted particular attention for their potential therapeutic use. However, an efficient clinical application requires a thorough knowledge of the biological mechanisms governing self-renewal and differentiation of MSCs, not yet achieved.
Recent studies underline the importance of a common set of key genes involved in the regulation of multiple tissues. One of them is Kit, that encodes for the tyrosine-kinase receptor of Stem Cell Factor (SCF) and has fundamental roles in different types of stem cells, such as Primordial Germ Cells (PGCs), cardiac stem cells, hematopoietic stem and progenitors cells, melanoblasts and mesangioblasts. However its involvement in MSCs is still controversial. In the laboratory where I carried out my graduation thesis, one of the projects is aimed at investigating the possible role of Kit in MSCs. Preliminary results have shown that Kit is transcriptionally active in undifferentiated MSCs of both mouse and human and its expression is modulated during their differentiation.
The aim of my work has been to analyze Kit expression at the protein level, in order to confirm its activity in murine MSCs, particularly during their adipogenic differentiation. For this purpose, using antibodies against the Kit extra-cellular domain, I performed Western Blot and immunocitochemestry assays as well as FACS analysis (Fluorescence Actived Cell Sorting). The results obtained by the different techniques have shown a low Kit expression in undifferentiated MSCs and an increase of the protein levels in the first steps of the adipogenic differentiation, followed by a return to basal levels in the terminal stages. Furthermore, in our laboratory, it has been characterized a transgenic mouse Kit-GFP in which the gene for the
Abstract
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Previous experiments have indicated that the transgene, active in different cellular types where the endogenous Kit is also active, faithfully recapitulates Kit expression. Therefore, this transgenic mouse represents a good model to study Kit activity in different tissues. In this line, using either fluorescence microscope or FACS analysis, I have monitored Kit-GFP MSCs during their differentiation along the adipogenic lineage. Such analyses have revealed an increased expression of the transgene, confirming the previous findings on the endogenous Kit.
In conclusion, my results highlight that Kit is active also in the MSCs, especially in the first stages of in vitro adipogenesis. In addition, the data suggest that the transgenic line contains the regulatory elements necessary for its correct expression also in the MSCs.
This finding provides the first evidence that Kit may contribute to the control of the adipose tissue too.
