RESUMO
Purpose: To investigate the level of cellular senescence in stem cells derived from microfragmented abdominal adipose tissue harvested from patients with knee osteoarthritis (OA). Methods: Stem cells harvested from microfragmented abdominal adipose tissue from 20 patients with knee OA, aged 29-65 years (mean = 49.8, SD = 9.58), were analysed as a function of patient age and compared with 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 ß-galactosidase activity were measured by flow cytometry. Cellular proliferation was assessed using a 5-ethynyl-2'-deoxyuridine proliferation assay. Stemness was assessed by stem cell surface markers using flow cytometry and the capacity to undergo adipogenic and osteogenic differentiation in vitro. 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. Conclusions: Autologous microfragmented adipose tissue-derived stem cells may be used in clinical trials of knee OA of patients aged 29-65 years, at least until passage 4, as they show stemness potential and negligible senescence in vitro. Level of Evidence: Not applicable.
RESUMO
PURPOSE: To investigate if viable stem cells could be isolated and expanded from cryopreserved microfragmented adipose tissue (AT) harvested from patients with knee osteoarthritis. METHODS: Microfragmented abdominal AT from knee osteoarthritis patients was cryopreserved at -80 °C in cryoprotectant-medium. The samples were thawed for stem cell isolation by tissue explant culture (TEC) and enzymatic digestion (ED), respectively. Viability, population doublings, and doubling time were assessed by trypan blue staining and flow cytometry. Cell type and senescence-associated ß-galactosidase activity were analyzed by flow cytometry. Osteogenic and adipogenic differentiation was assessed quantitatively by Alizarin-Red-S and Oil-Red-O staining, respectively. RESULTS: Microfragmented AT from 7 patients was cryopreserved for a period of 46-150 days (mean (SD) 115.9 days (44.3 days)). Viable stem cells were successfully recovered and expanded from all patients using both isolation methods with no significant difference in viable population doublings or doubling time from passage 1 to 3 (p > 0.05). Low levels of senescence-associated ß-galactosidase activity was detected for both methods with no significant difference between TEC and ED (p = 0.17). Stemness was verified by stem cell surface markers and osteogenic and adipogenic differentiation performance. Adventitial stem cells (CD31-CD34+CD45-CD90+CD146-), pericytes (CD31-CD34-CD45-CD90+CD146+), transitional pericytes (CD31-CD34+CD45-CD90+CD146+), and CD271+ stem cells (CD31-CD45-CD90+CD271+) were identified using both methods. More pericytes were present when using TEC (25% (24%)) compared to ED (3% (2%)) at passage 4 (p = 0.04). CONCLUSIONS: Viable stem cells can be isolated and expanded from cryopreserved microfragmented AT using both TEC and ED. TEC provides more clinically relevant pericytes than ED.
