Immunologic and genetic characterization of clinical-grade human MSCs. (A-C) Immunologic properties of 2 representative MSC productions obtained from donors 10 and 11 using FCS/FGF-2 were evaluated at the end of P1. (A) HLA-DR expression was quantified by flow cytometry. The percentage of positive cells and the rMFI (mean fluorescence intensity of HLA-DR/mean fluorescence intensity of Ig specific control) are indicated on the top right of each panel. (B) Responding PBMCs (105/well) were stimulated both with allogeneic MSCs from donors 10A and 11A (105/well) and with allogeneic mature dendritic cells (allo-DC, 104/well), 0.5 μg/mL pokeweed mitogen (PWM), and γ-irradiated PBMCs from the MSC donor when available (105/well) as positive controls. Each experiment was performed in sextuplicate culture wells. After 5 days of culture, cells were pulsed with tritiated thymidine (3H-TdR) for the last 16 hours, harvested, and counted on a liquid scintillation analyzer. (C) Responding PBMCs (105/well) were stimulated for 5 days with irradiated stimulator allogeneic PBMCs (105/well) in the absence (MLR) or presence of decreasing number of third-party MSCs. Each experiment was performed in sextuplicate culture wells. Cells were pulsed with tritiated thymidine (3H-TdR) for the last 16 hours, harvested, and counted on a liquid scintillation analyzer. (D) Growth profile of MSCs that exhibited (gray line) or not (dark line) karyotypic abnormalities. (E) Quantitative analyses of the expression of p53, p21, and c-myc cell-cycle regulators along passages. Each result was normalized to ABL and compared with expression levels in P2 MSCs. The arbitrary value of 1 was assigned to P2 MSCs. (F) SA-β-Gal staining of MSCs, which exhibited (12A2) or not (11A) karyotypic abnormalities, was performed at early (P3 and P2, respectively) and late (P13 and P7, respectively) passages. The percentages of positive cells are indicated on the top left of each panel. Original magnification ×10. (G) Culture in soft agar was performed at P7 for 2 MSCs that exhibited karyotypic abnormalities. HeLa cell line was used as a positive control. Original magnification ×10. Image acquisition details: Nikon Eclipse TE2000-S microscope, 10×/0.25 numeric aperture objective lens, Nikon DXM1200F digital camera, Lucia Version 5.00 software.
