Roles of intrinsic cell surface complement regulators in protecting MSCs after their contact with serum. (A) MSCs constitutively express all of the intrinsic cell-surface complement regulators. MSCs (5 × 105) were stained with 5 μg/mL of anti-CD55 IgG, anti-CD46 IgG, anti-CD59 IgG (solid lines), or isotype controls (dotted line) for 30 minutes on ice, then analyzed by flow cytometry. (B) Respective protective roles of these complement regulators. MSCs were incubated 10 μg/mL of control IgG, anti-CD55 IgG, anti-CD46 IgG, anti-CD59 IgG, or all of the IgGs, then subjected to the same BCECF leakage-based cytotoxicity assays. Representative results of 3 individual experiments. Data are mean ± SD, *P < .05. (C-D) Up-regulating human CD55 expression reduces MSC cytotoxicity after their contact with human serum in vitro. Eighty percent confluent MSCs in a well of a 6-well plate were transfected with CD55-expressing adenovirus or the same titer of empty control virus. In 48 hours, transfection efficiency was monitored by checking GFP expression under a fluorescence microscope (not shown), and levels of CD55 on MSCs cell surface was assessed by flow cytometry. (C) CD55 levels on different MSCs. Shaded area, isotype control; dotted line, MSCs without transfection; thin line, MSCs transfected with control virus; thick line, MSCs transfected with CD55-expressing virus. (D) Five × 105 of each MSCs without transfection, MSCs transfected with control virus, and MSCs transfected with CD55-expressing virus were subjected to the same BCECF leakage-based cytotoxicity assays. (E) Up-regulating mouse CD55 expression reduces MSCs cytotoxicity in vivo after infusion (n = 3 in each group, *P < .05). Representative results of 3 individual experiments. Data are mean ± SD, *P < .05.
