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Mesenchymal stem cells from human proximal femurs possess immunosuppressive activity


Journal title : National Medical Journal of China
Volume : 85
Issue : 39
Pagination : 2780-2784
Publication date : 01/12/2005

Summary

Objective: To evaluate whether mesenchymal stem cells (MSCs) obtained from human proximal femurs possess immunosuppressive effect so as to look for ideal bank of MSCs for clinical prophylaxis and treatment of graft versus host disease (GVHD). Methods: Human marrows were collected from the proximal femurs of patients undergoing hip replacement to isolate MSCs. The puncture materials obtained from the iliac bone marrow of 12 healthy donors were used as controls. Peripheral blood lymphocytes (PBLs) were obtained from the peripheral blood of healthy persons. 1 × 105 PBLs were mixed with allogeneic PBLs radiated by 60 cobalt and put into the wells of a 96-well plate. MSCs of the concentrations of 1 × 103, 3 × 104, 1 × 104, and 3 × 103, were added into the culture fluid of the mixed PBLs to be co-cultivated for 5 days. 1 μCi/well [ 3H]TdR was added in the last 18 hours. The cells were collected and the counts per minute (cpm) was detected. 3 × 104 and 1 × 104 MSCs were put into the wells. When the MSCs adhered to the wall, a membrane with micropores was inserted value of 1 × 105 PBLs and radiated allo-PBLs were put onto the top of which. Five days after cultivation 1 μCi/well [3H] TdR was added and the cpm was tested. MSCs were cultured in RPMI-1640 culture fluid and then contacted MLR constructed by 1 × 105 PBLs and 1 × 105 allo-PBL directly or via Transwell membrane with micropores. Five days after the supernatant was collected. ELISA was used to detect the content of TGF-β1, 0.1 μg/ml, 1 μg/ml, or 10 μg/ml anti-human TGF-β1 antibody was added to the co-cultivation system. Five days after [3H] TdR was added so as to test the value cpm. Results: 3 × 103-1 × 10 5 MSCs from proximal femurs inhibited the PBLs proliferation to 62 ± 18% - 28 ± 12% of maximal response, however, not significantly different from that observed in MSCs colleted from bone marrow of healthy donors via iliac crest aspiration (58 ± 12% - 27 ± 6%, P > 0.05). When these cells were separated physically from MLR system via a membrane with micropores 0.2 μm in diameter, 3 × 104 and 1 × 10 4 cells still markedly suppressed the PBLs proliferation to 36 ± 8% and 53 ± 13% of maximal response. ELISA showed that TGF-β1 was measured in the supernatants of MSCs, MSCs + MLR and MSCs + MLR in Transwell system, without significant differences among these experimental conditions. Furthermore, the presence of increasing amounts of neutralizing anti- TGF-β1 antibody did not reverse the inhibitory effect. Conclusion: MSCs obtained from the proximal femurs possess immunosuppressive activity. A soluble factor/factors was/were involved in such effect, however, TGF-β1 was not a candidate

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