MSCs are injured by complement after their contact with serum. (A) MSCs are injured after incubation with serum. Five × 105 of MSCs from different health donors (707, 725, 745, 751,731,741,742,749, and T) were first labeled with BCECF, then incubated with 20μL and 30 μL NHS in 100 μL of GVB++ buffer at 37°C for 30 minutes. MSCs cytotoxicity was assessed by measuring BCECF leaked into the supernatants. Representative results of 3 individual experiments. Data are mean ± SD, *P < .05. (B) Complement activation product C3b detection on MSC cel-surface. MSCs (1 ×106) were incubated with 30 μL of NHS in 100 μL of GVB++ buffer either without or with 1μM of EDTA (to inhibit complement activation). After incubation at 37°C for 30 minutes, the washed cells were stained with 5 μg/mL of FITC-labeled anti–human C3b mAb, and analyzed by a flow cytometer (LSR II). Representative results of 3 individual experiments. (C) Role of complement in the serum-mediated MSCs cytotoxicity (in vitro). Five × 105 of BCECF-labeled MSCs were incubated with 10, 20, or 30 μL of NHS or C3–depleted sera (C3-Depl) in 100 μL of GVB++ buffer. After incubation at 37°C for 30 minutes, MSCs cytotoxicity was assessed by measuring BCECF leaked into the supernatants. Representative results of 3 individual experiments. Data are mean ± SD, *P < .05. (D-E) Role of complement in serum-mediated MSCs cytotoxicity after infusion (in vivo). 1.0 × 106 BCECF-labeled MSCs were adoptively transferred into WT, C3−/− (D) and complement-depleted mice (E) by tail vein intravenous injection, serum levels of leaked BCECF in these mice were measured 20, 40, and 60 minutes after the injection. n = 3 in each group, data are mean ± SD, *P < .001.
