150 M. François et al. Figure 2. Protein and mRNA expression level of freshly thawed human MSC compared with MSC in culture. (A) IDO protein expression was analyzed by immunoblot on two human MSC donors freshly thawed and cultured for 1 and 7 days. IDO protein expression was induced by stimulating the MSC with 5 ng/mL recombinant (rh)IFN-γ for 24 h. Protein expression of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (B) Phosphorylated STAT-1 protein expression was analyzed by immunoblot on two human MSC donors freshly thawed and cultured for 1 and 7 days. STAT-1 phosphorylation was induced by stimulating the MSC with 5 ng/mL IFN-γ for 20 min. Protein expression of total STAT-1 was used as a loading control. (C) mRNA expression levels of (i) IDO, (ii) CCL2 and (iii) IL6 were measured by real-time qPCR in two MSC donors freshly thawed and cultured for 7 days. MSC were left untreated or stimulated with 5 ng/mL rhIFN-γ for 24 h. (D) mRNA expression level of Hsp27, Hsp47, Hsp56, Hsp70A, Hsp70B and Hsp90 by real-time qPCR performed on three MSC donors immediately after thawing and following a post-thaw recovery period of 4, 8, 16 and 24 h. Ribosomal 18S RNA was used as an internal control. Figures show representative results with means SD. affected by IFN-γ stimulation (Figure 2Ciii). These results suggest that transcript levels of constitutively produced proteins are not altered while transcrip- tion of IFN-γ-inducible genes is. Studies performed on human ﬁbroblasts have demonstrated that cryo- preservation and thawing induces the expression of heat-shock proteins (Hsp) Hsp27, Hsp47, Hsp56 and Hsp90, which diminishes within 24 h (12). In addition, Hsp27 has been demonstrated to inhibit cellular protein synthesis by blocking the assembly of the translation cap initiation complex (13). In order to determine whether the transcription of Hsp genes is altered by stress induced by the cryopreservation and thawing process in MSC, we performed real-time