Inflammatory stimulation with IL-1β and TNF-α alters the secretome of unsorted and sorted CD34+, CD146+, and CD271+ stromal/stem cells from microfragmented adipose tissue of osteoarthritis patients

Treatment of osteoarthritis (OA) with microfragmented adipose tissue (MFAT) has shown promising, but varying results. Stem cell populations expressing either CD34 + , CD146 + , or CD271 + markers are present in MFAT with considerable variability among patients. Paracrine signaling drives the therapeutic activity of transplanted stem cells through immunomodulation and the activation of local progenitor cells to improve joint health. However, non-sorted stem cells have been reported to acquire a pro-inflammatory secretome under OA-related inflammatory conditions, which may lower their therapeutic performance. This study aimed to compare the secretome of CD34 + , CD146 + , and CD271 + MFAT-derived stromal/stem cells to identify the cell type(s) that produce a secretome of particular therapeutic relevance for OA treatment when exposed to OA-like inflammation. CD34+, CD146+, and CD271+ stromal/stem cells were enriched from MFAT of patients with knee OA ( n = 8 patients, N = 29 cell lines) using magnetic-activated cell sorting (MACS) and analyzed as distinct subtypes. Unsorted cells were used as a control. The cells were treated with IL-1β and TNF-α or cultured without inflammatory factors for 48 hours. Cellular morphology was assessed microscopically. Cells secreting anti-inflammatory IL-10 were analyzed using flow cytometry. A multiplex secretome analysis of 46 growth factors and pro- and anti-inflammatory cytokines was performed using the Luminex® platform. Secretome analysis of pro-chondrogenic TGF-β1 was measured by Ella TM immunoassay. Data normality was assessed, and statistics were performed using either a mixed-effects model or multiple Wilcoxon matched-pairs signed rank tests with post hoc corrections. p -values < 0.05 were considered statistically significant. Enrichment of CD34, CD146, and CD271 stromal/stem cells from MFAT achieved a two-fold increase using MACS. The majority of both unsorted and sorted CD34, CD146, and CD271 cell populations secreted the anti-inflammatory cytokine IL-10. All analyzed MFAT-derived stromal/stem cell types displayed morphological changes and secreted growth factors associated with joint regeneration, together with both anti-inflammatory and pro-inflammatory cytokines with an upregulation after exposure to IL-1β and TNF-α. No major differences were observed between cell types. Fewer than 4 of 46 analytes differed significantly among cell types. 62% of the identified upregulated cytokines, in all four cell types, had a pro-inflammatory profile after IL-1β and TNF-α stimulation. None of the secretomes from the sorted stem cell types seemed superior to one another or to the secretome of unsorted MFAT-derived stromal/stem cells. Following stimulation with IL-1β and TNF-α, both unsorted and sorted CD34 + , CD146 + , and CD271 + MFAT-derived stromal/stem cells from OA patients exhibited an upregulated secretion of growth factors, as well as and pro- and anti-inflammatory cytokines. No distinct benefit of cell sorting was observed regarding paracrine function under OA-like inflammatory conditions based on the parameters assessed.