Influence of In Vitro Cultivation on Differentiation Gene Expressions in Canine Adipose-Derived Mesenchymal Stem Cells

Canine adipose-derived mesenchymal stem cells (cAD-MSCs) have been demonstrated to be effective in treating several illnesses due to, among others, their differentiation (DI) potential. However, some treatments require in vitro multiplication of cAD-MSCs, which may alter their DI characteristics. This research aimed to determine if in vitro cultivation affects DI processes in the molecular profiles of cAD-MSCs. Isolation of cAD-MSCs from the abdominal adipose tissue of eight young female dogs was performed, and their immunophenotype and in vitro DI potential was confirmed. Total RNA was extracted in the early passage (P) 3 and late P6, and changes in the gene expression profile linked to DI were examined using a microarray. Isolated cAD-MSCs arrested proliferation at P8, on average. They displayed typical immunophenotypes and maintained their DI potential during in vitro cultivation. Gene up- and downregulations were observed; however, the overall expression was not significantly altered comparing P3 to P6. The obtained results of up- and downregulations suggest in vitro cultivation may favour DI events, as indicated by the overexpression of the Growth DI factor 7 (GDF7), a crucial gene for cartilage formation, Kinase insert domain receptor (KDR), a protein involved in angiogenesis during DI, and Fibroblast growth factor 10 (FGF10), a protein required for the development of embryonic limbs. However, T-box transcription factor (TBX5), a promoter of osteoDI and mineralization, was downregulated in P6, suggesting prolonged in vitro cultivation could influence the osteoDI potential. The downregulation of Ras Homolog Family Member (RHOA) could be advantageous since overexpression of this gene is linked to the proliferation and spread of malignant cells. More donors and a comparison of the genders should be included in future research. Thus, this research demonstrated that, despite minor alterations, in vitro multiplication did not significantly alter the DI potential and gene expression of young female cAD-MSCs, which is a vital prerequisite for producing high-quality canine stem cell products.