148 M. François et al. Methods Cells Human MSC were isolated and cultured as described elsewhere (9). After the third passage, the MSC cultures were assayed by ﬂow cytometry analysis for the absence of CD45 and CD31 contaminating cells and expression of CD44, CD73, CD90 and CD105 (BD Bioscience, Mississauga, ON, Canada), and subjected to a differentiation assay toward adipocyte and osteocyte lineages, as described elsewhere (10). Human peripheral blood mononuclear cells (PBMC) were isolated from leu- kapheresis obtained from healthy volunteers using a Ficoll density gradient. Cell viability Frozen MSC were thawed, washed in media and cultured for 4 h, 24 h or 7 days before all cells, ﬂoat- ing and adherent, were analyzed using an annexin V and propidium iodide (PI) staining kit (Invitrogen, Burlington, ON, Canada) and quantiﬁed by ﬂow cytometry. Trypan blue exclusion staining was also carried out in parallel, and a consistently greater ‘viability’ assessment was observed compared with annexinV/PI staining (trypan blue viability 80%, annexinV/PI viability 60%, immediately after thawing). We believe this is due to the fact that trypan blue staining only shows dead cells and does not include dying cells; therefore trypan blue values were disregarded. T cell proliferation assay Carboxyﬂuorescein succinimidyl ester (CFSE; Invitrogen)-labeled PBMC were cultured at 400 000 cells/well in a 24-well plate with cultured, freshly thawed or paraformaldehyde (PFA)-ﬁxed MSC at a ratio of MSC:PBMC of 1:3 and 1:9. T lymphocytes were stimulated using 0.4 μg/mL anti- human CD3 and CD28 antibodies (clone HIT3a and CD28.2, respectively, from BD Pharmagen, Mississauga, ON, Canada). T-cell proliferation was determined 4 days later by ﬂow cytometry analysis of CFSE ﬂuorescence intensity. Western blot Anti-human IDO (GenScript, Piscataway, NJ, USA) and anti-human phospho-STAT-1 (clone 58D6; Cell Signaling, Pickering, ON, Canada) -speciﬁc antibod- ies were used to detect protein expression on whole cell protein extracts isolated from MSC by immuno- blot analysis. Where indicated, MSC were activated with recombinant human IFN-γ (5 ng/mL; R&D System, Burlington, ON, Canada). Real-time quantitative polymerase chain reaction DNA-free total RNA was extracted and reverse tran- scribed as described elsewhere (10). Real-time quan- titative polymerase chain reaction (qPCR) assays were performed in duplicate on an ABI 7500 fast real-time PCR system thermal cycler and SYBR green master- mix (Applied Biosystems, Carlsbad, CA, USA) with human primer sequences (5′3′ forward, reverse) for: IDO,GCCCTTCAAGTGTTTCACCAA,CCAGCC AGACAAATATATGCGA; Chemokine (C-C motif) ligand 2 (CCL2), CAAGCAGAAGTGGGTTCAG- GAT,TCTTCGGAGTTTGGGTTTGC;interleukin (IL-6), ATGAAGTTCCTCTCTGCAAGAGACT, CACTAGGTTTGCCGAGTAGATCTC; heat shock protein (Hsp27), CTGGCGCGTGTCCCTGGATG, GCTAGCTTGGGCATGGGGGC; Hsp47, CTGC TAGTCAACGCCATGTTCTTC, TCTGTGTCCA ACTCAAAGGC; Hsp56, TCCTCACCCCCGACG- GTGTG, CTGGCTTGTGCTGCCTGGCT; Hsp 70A,GGTCCGGATAACGGCTAGCCT,CCACCG GGTCGCCGAACTTG; Hsp70B, CATGCCCCG ATCTGCCCGAAC, GCACCTTCCCGCCCAGT TGAG; Hsp90, ATGGCTGCTTCCCAGGTGAT GCC, ACCTCAGGCTCATGACACCAGC; 18S ribosomal RNA (18S), TTACCAAAAGTGGCCCA CTA, GAAAGATGGTGAACTATGCC. Primers were designed using the National Center for Biotech- nology Information (NCBI)/primer blast designing tool. Data were analyzed using the relative quantiﬁ- cation method (11). Statistical analysis Data are reported as mean standard deviation (SD). All calculations were carried out using Graph- Pad Prism software (GraphPad Software, La Jolla, CA, USA). Comparisons between groups were made by ANOVA. Results and discussion To determine the immunosuppressive potential of cryopreserved MSC toward T-cell activation, we ana- lyzed their potency to suppress PBMC proliferation in comparison with MSC maintained in culture for 7 days. Early passage MSC were frozen in α Mini- mum Essential Medium (MEM), 30% fetal bovine serum and 5% dimethyl sulfoxide at –80°C for 24 h, then transferred to liquid nitrogen for 1 week before use. Cell viability analysis by ﬂow cytometry using annexin V- and PI-labeling revealed that, in cultured MSC, the percentage of live cells (annexin V– and PI–) was 92% and 91% for donors A and B, respectively, while in their thawed counterparts the percentage of live cells dropped to 61% and 44%, respectively (Figure 1A). We next tested post-thaw