Figure 1. Immunosuppressive potential of freshly thawed human MSC compared with MSC in culture. (A) Cell viability analysis was performed on freshly thawed and cultured MSC using annexin V and PI labeling. (B) T-cell proliferation assays were performed using CFSE-labeled human PBMC activated with 0.4 μg/mL anti- CD3 and -CD28 antibodies and co-cultured for 4 days with or without MSC maintained in culture for 7 days or freshly thawed at a MSC:PBMC ratio of 1:3. Cell proliferation was determined by ﬂow cytometry after gating lymphocytes on the forward and side scatter plot and measuring the percentage of CFSElow T cells. (C) T-cell proliferation assay performed as in (B) using cultured MSC mixed with PFA-ﬁxed MSC at a ratio of live:ﬁxed of 2:1, 1:1, 1:2 or 100% ﬁxed. A MSC:PBMC ratio of 1:3 was used. (D) T-cell proliferation assays performed as in (B) on two MSC donors freshly thawed and cultured for 1 and 7 days. Figures show representative results with means SD. cell culture restoration of immune suppressor func- tion. Freshly thawed cryopreserved MSC and MSC in the log phase of growth were co-cultured with CFSE-labeled PBMC activated with anti-CD3 and -CD28 antibodies at a MSC:PBMC ratio of 1:3, and T-cell proliferation was measured 4 days later. Cryo- preserved MSC had a signiﬁcantly impaired inhibi- tory effect on the proliferation of activated T cells Effect of cryopreservation of human MSC 149 in comparison with their cultured counterpart maintained in the log phase of growth (P 0.0001) (Figure 1B). T-cell hyperproliferation observed in the presence of thawed MSC suggested that dead MSC found in the freshly thawed MSC samples may activate T-cell proliferation. To test this hypothesis, we performed a T-cell proliferation assay in which cultured MSC were mixed at a different ratio with PFA-ﬁxed MSC, which served as dead cells, while the MSC:PBMC ratio remained 1:3 (Figure 1C). Adding increasing amounts of ﬁxed MSC while reducing the amount of live MSC diminished the level of T-cell immunosuppression. Hyper-proliferation of T cells could also be observed at ratios of 1:1, 1:2 and 100% ﬁxed, depending on the MSC donor, which may have been caused by the low amount of IDO produced in comparison with the pro-inﬂammatory cytokines [i.e. interleukin (IL)-6] produced by live MSC. Four hours of adherent or suspension culture of MSC after thaw- ing were not sufﬁcient to restore their immunosuppres- sive capabilities (data not shown). However, a culture period of 24 h was sufﬁcient to restore the immunosup- pressive properties of thawed MSC to a level similar to that of MSC cultured for 7 days (Figure 1D). IFN-γ-inducible expression of IDO in human MSC has been identiﬁed as one of the main mecha- nisms of T-cell immunosuppression (2). We therefore tested the induction of IDO protein expression in MSC. Freshly thawed MSC stimulated with IFN-γ showed very low levels of IDO protein expression compared with cultured MSC, while culture recov- ery for at least 24 h restored IFN-γ responsiveness and IDO induction (Figure 2A). This observation suggests that the MSC biochemical response to IFN-γ and/or the protein synthesis machinery is compromised. To test this hypothesis, we analyzed Signal Transducers and Activators of Transcription (STAT-1) phosphorylation in response to IFN-γ stimulation by immunoblotting, and found that phosphorylated STAT-1 was markedly diminished in thawed compared with cultured MSC (Figure 2B). Consistent with these data, we queried the mRNA transcript levels of IDO, and of two constitutively expressed genes, CCL2 and IL-6. No basal expres- sion mRNA encoding of IDO was detected, while the basal levels of CCL2 and IL-6 mRNA gene expression were comparable in freshly thawed and cultured MSC (Figure 2C). In response to IFN-γ, the up-regulation of IDO and CCL2 mRNA expres- sion was signiﬁcantly diminished in thawed MSC. In two individual donors, IFN-γ-induced levels of IDO mRNA decreased 2.7- and 5-fold (P 0.001; Figure 2Ci), while CCL2 mRNA expression decreased 4- and 7-fold (P 0.001; Figure 2Cii). IL-6 regulation is mainly regulated by tumor necro- sis factor (TNF)-α, which appeared only slightly