Figures & data
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.
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.
