qPCR on MSC samples immediately after thawing and following a post-thaw culture recovery period of 4, 8, 16 and 24 h. For this assay¸ primer sequences for Hsp27, Hsp47, Hsp56, Hsp70A, Hsp70B and Hsp90 were tested. We observed an up-regulation of mRNA level of Hsp70A and Hsp70B as early as 4 h post- thaw, which peaked at 8 h post-thaw and returned to basal levels within 24 h for three individual MSC donors tested (Figure 2D). These data support the theory that thawed MSC recapitulate a molecular genetic heat-shock response that reverses within 24 h, and provides a mechanistic rationale for the loss of IFN-γ responsiveness and blunted suppres- sive licensing (14). Therefore, we speculate that the stress response induced by cryopreservation recovery may temporarily prevent MSC from responding to immune cues and secondary suppressor functions. In conclusion, our data demonstrate that cryo- preservation negatively affects the immunosuppres- sive properties of MSC in a reversible manner, and is associated with a heat-shock stress response initiated during the thawing process. A culture recovery period of at least 24 h is able to restore the immunosup- pressive properties of MSC, including transcriptional IDO responsiveness to IFN-γ and down-regulation of Hsp expression. In addition, phosphatidylser- ine translocation to the cell surface is a reversible process and not a strict commitment to apoptosis (15,16). Temporary repression of non-constitutively expressed genes during the stress response allows the cells to prioritize cell survival before recover- ing their functional properties (14). Allowing MSC to recover for a period as short as 24 h post-thaw may reverse early apoptosis, initiate internalization of phosphatidylserine, and possibly prevent in vivo clearance of annexin V-positive MSC by phagocytes (17,18), in addition to down-regulating the cellu- lar stress response. We propose that the potency of MSC as a suppressor therapy for auto/alloimmune ailments may be improved if delivered in a metaboli- cally optimal condition. Acknowledgments MF was supported by the F. Banting and C. Best Doctoral Bursary of the Canadian Institute of Health Research (CIHR). This work was funded by CIHR operating grant MOP15017 and a Georgia Cancer Coalition Award to JG. Authorship contributions: MF designed and per- formed research, analyzed and interpreted data, and wrote the manuscript; IBC and RRM designed experiments; SY performed experiments; EKW interpreted data and edited the manuscript; JG designed the research plan and is senior author. 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