ABSTRACT
Mesenchymal stem cells are clonogenic multipotent cells, able to differentiate into multiple mesodermic cell types, such as ostebloasts, condrocytes, adipocytes, miocytes and stromal cells. Mainly localized in the bone marrow, they have been isolated also from other tissues, such as skeletal muscle, adipose tissue, amniotic fluid, teeth, cord blood, ect. MSCs have raised to a great interest because of their easy accessibility and their ability to be grown and differentiated in vitro. Moreover, for their biological properties, MSCs represent a potentially precious therapeutic tool in regenerative medicine for the treatment of many deseases. Their optimal use depends on the knowledge of the molecular mechanisms that control their expansion and differentiation, however, so far, aspect of their biology are still unkonwn.
Kit is a gene encoding the tyrosine-kinase receptor for the Stem Cell Factor (SCF). It is
expressed in many different stem and progenitor cell types, such as primordial germ cells, hematopoietic stem and progenitor cells, cardiac stem cells, melanoblasts, intestinal Cajal cells, ect. The role of this gene in MSCs is still controversial and the aim of my thesis has been to study Kit espression in human and murine MSCs, in an undifferentiated state and after induction toward an osteogenic or an adipogenic fate.
Primary cultures of human and murine MSCs, derived from bone marrow, have been expanded and differentiated in vitro with inducing agents for osteogenic (β- Glicerophosphate, Ascorbic Acid and Dexamethasone) or adipogenic differentiation (Dexamethasone, Indometacin, Isolbutilmetilxanthine and Insulin). The differentiation process was monitored by expression analysis of osteogenic (Runx2 and Alp) or adipogenic (PPARγ and C/EBPα ) markers, using Real-Time qPCR. In addition, cell differentiation was evaluated by cytochemical stainings, with Alizarin Red that binds the Calcium-Phophates of
the matrix secreted by mature osteoblasts, or with Oil Red that underlines the presence of the lipidic drops inside the adipocytes. Kit espression has been evaluated by Real-Time qPCR in undifferentiated cells and at different steps of the osteogenic and adipogenic differentiations.
Our results show that, both in human and murine undifferentiated MSCs, Kit is expressed at low level and during the osteogenic differentiation this gene is switched off. In contrast, during the adipogenic differentiation, Kit expression is up-regulated during early stages of the differentiation and then down-regulated at later stages, suggesting that this gene may likely play a role in the activation of the adipogenic program. To confirm this hypotesis, Kit espression has been thoroughly analysed in transgenic mice Kit/GFP, where the GFP gene is under the transcriptional control of Kit regulatory regions. Previous studies made in our laboratory have demonstrated that the expression of the transgene faithfully recapitulates the expression of the endogenous Kit. MSCs were obtained from Kit/GFP mice bone marrow, then expanded in vitro and differentiated along the osteogenic or the adipogenic lineage.
Expression of the Kit/GFP transgene was monitored by fluorescence analysis and Real-Time qPCR in undifferentiated cells as well as a different times after induction. Results show that the transgene is active already after 8 hours of treatment and it’s expression profile is very similar to the expression profile of the endogenous Kit gene. Therefore, these results represents a first evidence suggesting a possible role for Kit in adipogenesis. The comprehension of the molecular mechanisms that regulate adipogenesis may have important implications for the diagnosis or the treatment of many diseases of the adipose tissue.
