Replicative senescent MSCs display attenuated immunosuppressive and intact lung-homing properties. Replicative fit and senescent MSCs were cultured with αCD3αCD28 Dynabeads-stimulated PBMCs. Four days post culture, T-cell proliferation was measured by Ki67 intracellular staining. (A) Representative fluorescence-activated cell sorting (FACS) plot and (B) cumulative effect of fit and senescent MSCs’ (n = 5 donor pairs) effect on T-cell proliferation (% CD3⁺Ki67⁺) is shown. CFSE-labeled PBMCs were cocultured with replicative fit and senescent MSCs and were stimulated with αCD3αCD28 Dynabeads. On the fourth day, intracellular cytokine staining was performed to determine the percentage of cytokine-secreting proliferated T cells CD3⁺CFSE^dim IFNγ T cells. (C) Representative FACS plot and (D) dose-dependent effect are shown. Individual or mixed fit and senescent MSCs at 1:1 ratio were cultured with αCD3αCD28 Dynabeads^-stimulated PBMCs and 4 days post culture, T-cell proliferation was measured as indicated above. (E) Representative FACS plot and (F) cumulative effect are shown. Similar results were obtained in a repeat experiment with an additional 1 or 2 fit and senescent MSC donor pairs. (G) Fit or senescent MSCs (0.5 × 10⁶/animal) were injected IV into C57BL/B6 mice via the tail vein. At 24 hours post-infusion, the animals were killed and the lungs were excised to extract total gDNA for real-time PCR amplification of human gDNA and mouse gDNA. Human gDNA threshold cycle (CT) values were normalized with mouse gDNA values. Cumulative inverse CT values with mean ± SD were shown from 2 independent experiments (n = 6 animals per group), performed with 2 unique fit and senescent MSC donors pairs. P < .05 was considered statistically significant based on 2-tailed Student t tests.