In vitro production of differentiated Endotelual Cells from Mesenchimal Stem Cells, collected from adult rat Adipose Tissue (AD-MSCs): preliminary results

Mesenchymal Stem Cells (MSCs) are multipotent cells that reside within various tissues, including adipose tissue and bone marrow. Research concerning the potential therapeutic role of MSCs has advanced significantly over the last decade. Many studies have demonstrated that adipose tissue is an excellent source of MSCs (AD-MSCs), easily accessible in experimental animals. AD-MSCs can be expanded in vitro over the short term and they can differentiate toward a variety of cell types of mesodermal lineage. Therefore, they are thought an attractive tool for cell therapy. Selection of appropriate animal models for preclinical evaluations is crucial for optimization and validation of therapeutic protocols in regenerative medicine. Therefore, in vitro studies are necessary to future in vivo applications of MSCs. Rat is an animal model usefull for a large number of in vitro studies. Aim of this work is the in vitro production of endothelial cells differentiated from rat ADMSCs. For this purpose, subcutaneous adipose tissue samples were collected from 5 Wistar rats, in accordance to the European Directives (86/609/CEE and 63/2010/EU) concerning the protection of experimental animals. These samples were processed for in vitro AD-MSCs isolation. After 2 days, nonadherent cells were removed and medium (α-MEM) was replaced. The adherent AD-MSC population was expanded for 5 passages. To assess their characteristics, the expression of stemness markers, such as OCT4, SOX2 and NANOG, was evaluated by Real Time Sybr Green PCR. The analysis of gene expression was performed from 0 to 5 passage. For this purpose, mRNA was extracted from each passage using a commercial kit. Successively, mRNA was retrotranscribed in cDNA and stemness genes were detected using specific primers pairs, designed by the use of Primer Express software. Gene expression analysis was normalised by the GADPH gene housekeeping expression. To induce endothelial differentiation, stem cells at passage 0 and 1 were cultivated in D-MEM medium for 18-24 h. Successively, D-MEM medium was replaced with an enriched medium with VEGF-C. Cells were cultivated for 15 days. To verify the begin of differentiation, gene expression analysis for endothelial markers CD31, eNOS and vWF was performed by Real Time Sybr Green PCR. Specific primers pairs were designed by the use of Primers Express software. Gene expression results show an increase of expression levels for stemness markers, up to the in vitro passage 3. The begin of the endotelial differentiation is proved by the initial increase of expression levels for CD31, eNOS and vWF, associated to a decrease in the expression levels for stemness markers. These are preliminary results of a project aimed to the in vitro production of endotelial differentiated cells from AD-MSC. Further studies are required to confirm the acquisition of the endothelial phenotype by AD-MSC cells, induced to differentiate.