Abstract Mesenchymal stem cells present clinical potential to recover and regenerate injured tissues in diverse pathologies. The in vitro expansion and characterization of these cells contribute to elucidation of the mechanisms of senescence and strategies involving cell therapies. This study aimed to compare specific characteristics between initial and advanced passages of mesenchymal stem cells derived from adipose tissue and bone marrow. Both cell types were characterized according to immunophenotype, osteogenic differentiation, genomic instability, migration assay, doubling population time and colony forming ability. Our results demonstrated that both cell types were able to maintain an immunophenotypic profile typical of mesenchymal stem cells during increasing passages. Adipose stem cells at initial passage presented greater migration capacity compared to advanced passage cells, and advanced passage cells proliferated faster than initial passage cells. Bone marrow stem cells at early passages presented higher osteogenic potential than advanced. At advanced passages they presented higher colony forming capacity and genetic damage than those at initial passage. These results suggest that mesenchymal stem cells maintained in culture presented characteristics of senescence that should be monitored prior the use in regenerative medicine and cells derived from bone marrow at initial passage have better potential for therapeutic use in bone tissue engineering.