Characterization of Senescence of Human Adipose-Derived Stem Cells After Long-Term Expansion

Since the 1980s, adipose-derived stem cells (ASCs) have become a powerful and potential source for stem cell-based therapy, regenerative medicine, and even drug delivery in cancer treatment. The development of off-the-shelf mesenchymal stem cells (MSCs), including ASCs, has rapidly advanced in recent years with several clinical trials and approved products. In this technology, ASCs should be expanded long term in order to harvest higher cell number. In this study, senescence of ASCs after long-term expansion was evaluated.

Human ASCs (hASCs) were isolated and cultured continuously at a density of 10³ cells/cm² up to passage 15. The cells were assessed for aging via changes in the following: characteristics of MSCs, mitochondrial activity, accumulation of beta-galactosidase, and expression of tumor suppressor genes.

The results showed that following in vitro expansion to the 15th passage, ASCs did not show changes in immunophenotype, except for decreased expression of CD105. However, the cells increased in size and in shape and complexity (toward the "fried egg" morphology). They also almost ceased to proliferate in passage 15. Nonetheless, they maintained in vitro differentiation potential toward osteoblasts, chondrocytes, and adipocytes. Expression of tumor suppressor genes p53 and p16 did not significantly change, while p27 was significantly downregulated. Mitochondrial activities also decreased slightly in culture from passage 5 to passage 10 and remained stable to passage 15. ASCs also showed increased accumulation of beta-galactosidase in culture, but it was negligible.

In conclusion, hASCs exhibited some particular characteristics of aged stem cells when the number of subculture cells increased. However, up to passage 10, ASCs also retained almost all of the characteristics of MSCs.