Expression Profile of New Marker Genes Involved in Differentiation of Canine Adipose-Derived Stem Cells into Osteoblasts and Chondrocytes

Next-generation sequencing (RNAseq) analysis of gene expression changes during the long-term in vitro culture and osteogenic and chondrogenic differentiation of ADSCs remains to be important, as the analysis provides important clues toward employing stem cells as a therapeutic intervention. In this study, the cells were isolated from adipose tissue obtained during routine surgical procedures and subjected to 14-day in vitro culture and differentiation. The mRNA transcript levels were evaluated using the Illumina platform, resulting in the detection of 19,856 gene transcripts. The most differentially expressed genes (fold change >|2|, adjusted p value < 0.05), between day 1, day 14 and differentiated cell cultures were extracted and subjected to bioinformatical analysis based on the R programming language. The results of this study provide molecular insight into the processes that occur during long-term in vitro culture, osteogenic and chondrogenic differentiation of ADSCs, allowing the re-evaluation of the roles of some genes in MSC progression towards a range of lineages. The results improve the knowledge of the molecular mechanisms associated with long-term in vitro culture and differentiation of ADSCs, as well as providing a point of reference for potential in vivo and clinical studies regarding these cells' application in regenerative medicine. Overall design: To investigate the changes in gene expression after the osteogenic and chondrogenic differentiation and prolonged in vitro culture, we isolated mesenchymal stem cells from canine adipose tissue samples. We then performed gene expression analysis using the data obtained from RNA-seq of four group of samples: early controls, late controls cultured for 14 days, osteo- and chondro-induced ASCs cultured for 14 days. Each group of samples consisted of three biological replicates.