Differential secretome of activated PBMCs cocultured with fit and senescent MSCs. Replicative fit and senescent MSCs were cultured in the presence and absence of αCD3αCD28 Dynabeads-stimulated PBMCs in 2-chamber transwell plates. Four days post culture, T-cell proliferation was measured by Ki67 intracellular staining. (A) Relative effect of fit and senescent MSCs’ effect on T-cell proliferation (% CD3⁺Ki67⁺) in coculture and transwell culture are shown. Similar results were obtained in a repeat experiment with an additional 2 fit and senescent MSC donor pairs. (B) CD14⁺ purified macrophages were cultured on transwell and in the bottom PBMCs were activated with 2 independent fit or senescent MSC pairs. 48 hours later, macrophages in the transwell were harvested and IL-10 mRNA was measured with GAPDH mRNA as endogenous housekeeping control. Fold induction of IL-10 mRNA was derived through δ-δ CT method. Replicative fit and senescent MSCs were cultured in the presence and absence of αCD3αCD28 Dynabeads-stimulated PBMCs. Four days post culture, supernatant was collected and human cytokine 30-plex panel Luminex assays were performed according to the manufacturer’s instructions. Fit and senescent MSC pairs from 4 independent donors were tested against different PBMC donors. Cytokine levels were shown in pg/mL. F, fit MSCs and activated PBMCs; N, no MSCs and activated PBMCs; S, senescent MSCs and activated PBMCs. (C) IL-1ra, IFNγ, IL-2r, CCL4, TNFα, IL-5, and IL-10 were decreased efficiently by fit but not senescent MSCs. (D) VEGF, GCSF, CXCL10, and CCL2 were upregulated by fit MSCs cocultured with activated PBMCs and the senescent MSCs display attenuated upregulation. (E) FGF-basic was substantially increased by senescent MSCs compared with fit MSCs. (F) Control, CXCL9, CXCL10, CXCL11, GCSF, CCL2, and IDO siRNA-transfected MSCs were cultured with αCD3αCD28 Dynabeads-stimulated PBMCs in 2-chamber transwell plates for 3 to 4 days. Expression level of appropriate silenced mRNA relative to GAPDH was evaluated by quantitative SYBR green real-time PCR. δ-δ CT method was applied to calculate the fold induction of silenced gene over the control siRNA-transfected MSCs. (G) CXCL9, CXCL10, CXCL11, GCSF, CCL2, IDO, and control siRNA-transfected MSCs were cultured with αCD3αCD28 Dynabeads-stimulated PBMCs. Four days post culture, T-cell proliferation was measured by Ki67 intracellular staining. Proliferation of T cells (CD3⁺Ki67⁺) in the presence and absence of siRNA transfected MSCs is shown. Similar results were obtained in a repeat experiment with an additional independent MSC donor. (H) VEGF and FGF were added exogenously to fit MSCs cocultured with activated PBMCs at indicated ratios. Four days later, CD3⁺ T-cell proliferation was measured by Ki67 intracellular staining. Similar results were obtained in a repeat experiment with an additional independent MSC donor.