Expression of aquaporin-1 and cxcr4 in ovine mesenchymal stem cell.

Mesenchymal stem cells (MSCs) are a well characterized and highly adaptable cell source for regenerative medicine and tissue engineering. Ovine species is considered as a valuable model for human bone turnover and remodeling activity, due to the fact that adult animals show similar bone structure and composition [1]. Therefore, in orthopedic research, sheep are frequently employed for critical-size bone defects, which are then treated with different biomaterials combined with (predifferentiated) MSCs. Despite a considerable number of reports employing ovine MSCs (oMSCs) in tissue engineering, their molecular characterization is limited to few studies. Unlike human MSCs, ovine MSC are not well studied regarding isolation, expansion and surface antigen expression of oMSC. Recently, a family of 13 water-transporting membrane channel proteins, called aquaporins (AQPs) known for their key role in fluid transport in various epithelial and endothelial tissues, have been extensively studied [2]. Such proteins seem to have important functions in diverse biological processes related to cell migration, proliferation, apoptosis and cell volume-regulation. Furthermore, many studies have demonstrated that stem cell migration and organ-specific homing are regulated by chemokine and their receptors [3]. Therefore, the aim of this work was to determine whether water channel molecule, aquaporin 1 (AQP1) and chemokine receptor type 4 (CXCR4) could be expressed in o-MSCs to regulate some cellular events. MSC isolated from fresh bone marrow obtained from the posterior iliac crest of sheep were cultured on flasks by incubation in a humidified atmosphere at 37 °C with 5% CO2 in culture medium (a-MEM) supplemented with 10% fetal bovine serum (FBS). Further phenotypical characterization of MSC was performed via cytofluorimetric analysis of cell surface markers at passage 2. MSC resulted positive for CD44, CD73, CD90, CD105, CD146 and negative for CD34 and CD45. The analysis was performed using a FC500 flow cytometer (Beckman Coulter, Brea, CA, USA). To obtain conditionated Medium (CM), oMSCs were grown for different times 24 h, 48 h and 72 h in cultured medium with 1% FBS. Then, CM was collected, filtered and stored in aliquots at -20 °C until required, to investigate the impact of CM composition on AQP1 and CXCR4 expression. Morphological changes of cells exposed to CM were observed by light microscope. Whole protein extracts were prepared and analyzed by western blotting. Furthermore, oMSCs proliferation and migration analysis were performed also in presence of CM. Our results showed that oMSCs cultured in 10% FCS medium express high level of AQP1 and CXCR4. Furthermore, CM after an exposure to oMSCs for 48 h, causes an increase of AQP1 and CXCR4 expression as well as an enhancement of cell proliferation. In conclusion, the results of this study suggest that oMSCs produce some regulatory factors (cytokines, chemokines or growth factors) that promote their migration by autoctine pathways [4]. It is well known that these soluble mediators may act directly, triggering intracellular mechanisms of injured cells, or indirectly, inducing secretion of functionally active mediators by neighboring cells. For these reasons, the results of this study could improve the knowledge on oMSC characterization in consideration of their application in regenerative medicine because of handling and housing, cost, and ethical acceptance.