ABSTRACT
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 x 10(5) 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 x 10(5), 3 x 10(4), 1 x 10(4), and 3 x 10(3), were added into the culture fluid of the mixed PBLs to be co-cultivated for 5 days. 1 microCi/well [(3)H] TdR was added in the last 18 hours. The cells were collected and the counts per minute (cpm) was detected. 3 x 10(4) and 1 x 10(4) MSCs were put into the wells. When the MSCs adhered to the wall, a membrane with micropores was inserted value of 1 x 10(5) PBLs and radiated allo-PBLs were put onto the top of which. Five days after cultivation 1 microCi/well [(3)H] TdR was added and the cpm was tested. MSCs were cultured in RPMI-1640 culture fluid and then contacted MLR constructed by 1 x 10(5) PBLs and 1 x 10(5) 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-beta(1). 0.1 microg/ml, 1 microg/ml, or 10 microg/ml anti-human TGF-beta1 antibody was added to the co-cultivation system. Five days after [(3)H] TdR was added so as to test the value cpm. RESULTS: 3 x 10(3) - 1 x 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 collected 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 microm in diameter, 3 x 10(4) and 1 x 10(4) cells still markedly suppressed the PBLs proliferation to 36 +/- 8% and 53 +/- 13% of maximal response. ELISA showed that TGF-beta1 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-beta1 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-beta1 was not a candidate.