Fisetin Attenuates Cellular Senescence Accumulation During Culture Expansion of Human Adipose-Derived Stem Cells

Abstract Mesenchymal stem cells (MSCs) have long been viewed as a promising therapeutic for musculoskeletal repair. However, regulatory concerns including tumorgenicity, inconsistencies in preparation techniques, donor-to-donor variability, and the accumulation of senescence(definition) during culture expansion have hindered the clinical application of MSCs. Senescence, in particular, is a driving mechanism for MSC dysfunction with advancing age. Often characterized by increased reactive oxygen species, senescence-associated heterochromatin foci, inflammatory cytokine and chemokine secretion, and reduced proliferative capacity, senescence directly inhibits MSCs efficacy as a therapeutic for musculoskeletal regeneration and repair. Furthermore, autologous delivery of senescent MSCs can further induce disease and aging progression through the secretion of the senescence-associated secretory phenotype (SASP) and mitigate the regenerative potentetial of MSCs. To combat these issues, the use of senolytic agents to selectively clear senescent cell populations has become popular. However, their benefits to human MSCs during the culture expansion process have not yet been elucidated. To address this, analyzed markers of senescence during culturing of human primary adipose-derived stem cells (ADSCs), a population of fat-resident MSCs commonly used in regenerative medicine applications. Next, we used the senolytic agent fisetin to determine if we can reduce these markers of senescence within our culture-expanded ADSC populations. Our results indicate that ADSCs acquire common markers of cellular senescence including increased reactive oxygen species, senescence-associated β-galactosidase, and senescence-associated heterochromatin foci. Furthermore, we found that the senolytic agent fisetin works in a dose-dependent manner and selectively attenuates these markers of senescence while maintaining the differentiation potential of the expanded ADSCs. Signficance Statement The accumulation of dysfunctional, senescent cells throughout aging is not confined to specific tissues and cell types, but instead effects the whole body, including stem cells. Similarly, during culture expansion stem cells accumulate senescence while concurrently losing their regenerative potential. In this study, we found that fisetin (a well known senotherapeutic agent) can reduce the number of senescent cells during stem cell expansion. The current results indicate that fisetin may be used not only as a promising therapeutic to remove senescent cells in stem cell isolates from older individuals but also to reduce the accumulation of senescence during culture expansion.