Description
Objectives: Intra-articular injection of autologous stem cells from microfragmented adipose tissue is a promising treatment for patients with knee osteoarthritis (OA). However, studies on enzymatically processed adipose tissue have identified an increase in senescent stem cells with increasing donor age. This may diminish treatment outcome, due to lost proliferative and differentiation capacity, and secretion of inhibitory factors to surrounding cells. Our aim was therefore, to investigate the level of cellular senescence in stem cells derived from microfragmented adipose tissue harvested from patients with knee OA.
Methods: Stem cells harvested from microfragmented abdominal adipose tissue from 20 patients with knee OA, aged 29 to 65 years (mean = 49.8, SD = 9.58), were analyzed at passage 4 as a function of patient age, and compared with positive control cells exhibiting signs of cellular senescence. Steady state mRNA levels of a panel of genes associated with senescence were measured by qPCR. Intracellular senescence-associated proteins p16 and p21, and senescence-associated β-galactosidase activity were measured by flow cytometry. Cellular proliferation was assessed using a fluorescence 5-ethynyl-2’-deoxyuridine (EdU) proliferation assay. Stemness was assessed by stem cell surface markers using flow cytometry, and the capacity to undergo adipogenic and osteogenic differentiation in vitro. Statistical analyses were done using linear regression as a function of age and a Wilcoxon matched-pair signed mark test to assess differentiation. Normality of data was assessed by QQ-plots and Shapiro-Wilk tests. p-values <0.05 were considered statistically significant. Collection and molecular analyses of the cells were approved by the Danish National Committee on Health Research Ethics (H-18013145) and the Danish
Data Protection Agency (VD-2018-141).
Results: No correlation was found between cellular senescence levels of the microfragmented adipose tissue-derived stem cells and patient age for any of the standard assays used to quantify senescence. The level of cellular senescence was generally low across all senescence associated assays compared to the positive senescence control. Stemness was verified for all samples. An increased capacity to undergo adipogenic differentiation was shown with increasing patient age (p = 0.02). No effect of patient age was found for osteogenic differentiation performance.
Conclusions: Autologous microfragmented adipose tissue-derived stem cells may be used in clinical trials of knee OA of patients aged 29 to 65 years, at least until passage 4, as they show stemness potential and negligible senescence in vitro.
