Additional file 5: Figure S4. of A robust potency assay highlights significant donor variation of human mesenchymal stem/progenitor cell immune modulatory capacity and extended radio-resistance

MSPC immunosuppressive function is in part cell contact-dependent. Carboxyfluorescein (CFSE) prelabeled pooled peripheral blood mononuclear cells (pPBMC) were used after thawing in RPMI-1640/10 % pHPL in triplicate of 3 × 105 cells/well in the presence of phytohemagglutinin (+PHA; A, B, C; d4) or in the absence of PHA (D, E, F; d7). Mesenchymal stem/progenitor cells (MSPC) from three randomly selected bone marrow (BM, blue bars), white adipose tissue (WAT, yellow bars) and umbilical cord (UC, red bars) donors were added 24 hours in advance to the culture vessels. (A, B, C) PHA-induced mitogenesis in the absence (green bars) or presence of MSPCs in serial dilution as indicated was measured as percentage of proliferating CD3+ T cells. Inhibition of T-cell proliferation by decreasing numbers of MSPCs was determined in 96-well flat-bottomed plates in direct cell–cell contact between MSPCs and pPBMCs (left part of the graphs) as compared to cell contact-independent co-cultures with MSPCs in the lower compartment and CFSE-labeled pPBMCs in the transwell insert (grey area, right part of the graphs) using (A) BM-derived, (B) WAT-derived and (C) UC-derived MSPCs as regulatory cells in serial dilution as indicated by the MSPC:PBMC ratios on the x-axis indicating significant inhibition of T-cell proliferation by all three MSPC sources at most but not all ratios but significantly reduced in transwell assays for most ratios. (D, E, F) At day 7, the potent allogeneic MLR of the pPBMCs derived from pooling ten random donor-derived cells (grey bars) in the absence of external stimulation was significantly inhibited by all three MSPC sources at some but not all ratios tested. MSPCs were tested in direct cell–cell contact between MSPCs and pPBMCs (left part of the graphs) as compared to cell contact-independent co-cultures (transwell) as described above, accordingly. Significant difference are indicated (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n = 2). (DOC 259 kb)