Exogenous addition of kynurenine corrects senescence-associated impaired immunosuppressive properties of MSCs. (A) 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 kynurenine levels were performed according to the manufacturer’s instructions. Cumulative of 6 donor pairs were shown. (B) Catabolism of kynurenine pathway and the enzymes that facilitate this cascade is shown with a cartoon adapted from reference 30. IDO, kynurenine aminotransferases (KAT), kynurenine 3-monooxygenase (KMO), KYN, 3-hydroxyanthranilate 3,4-dioxygenase (HAAO), and quinolinate phosphoribosyl transferase (QPRT). Fit and senescent MSCs were cultured in the presence and absence of αCD3αCD28 Dynabeads-stimulated PBMCs in 2-chamber transwell plates. Four days later, MSCs were harvested and the expression level of mRNA of kynurenine catabolic enzymes relative to GAPDH was evaluated by quantitative SYBR green real-time PCR. (C) Representative heat map with CT values (red = high expression, blue = low expression) and (D) cumulative comparison between fit and senescent MSC pairs from 3 unique donors is shown. F+P, fit MSCs + activated PBMCs; S+P, senescent MSCs + activated PBMCs. (E) Control, KYN, KMO, 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. KYN, KMO, and 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. (F) 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. (G) Kynurenine (500 uM) was added exogenously in the coculture of αCD3αCD28 Dynabeads-stimulated PBMCs with and without fit and senescent MSCs. Four days post culture, T-cell proliferation was measured by Ki67 intracellular staining. F, fit MSCs + activated PBMCs; N, no MSCs + activated PBMCs; S, senescent MSCs + activated PBMCs. Kynurenine’s effect on T-cell proliferation with 4 independent fit and senescent MSC donor pairs is shown.