In vivo migration studies. Seven days after tumor cell inoculation into
the right frontal lobe, EGFP-expressing MSCs (MSCs-EGFP; 2 105) were
implanted into the opposite hemisphere (2 mm lateral and 1 mm anterior
to bregma, at a depthof 2.5 mm from the skull base), or PKH26 (Sigma)–
labeled MSCs-stTRAIL were implanted into the tumor mass. Migration
toward the tumor was assessed at 4, 7, or 10 d after MSCs inoculation
by direct visualization witha fluorescence microscope or a confocal
microscope (LSM 510 Meta, Carl Zeiss).

Assessment of cell viability and terminal deoxyribonucleotidyl
transferase–mediated dUTP nick end labeling staining. UCB-MSCs or
U-87MG cells (5 104) were seeded in 24-well plates, and increasing
amounts of Ad-stTRAIL or recombinant human TRAIL (rhTRAIL; R&D
Systems) were added to confirm TRAIL tumor-specific cytotoxicity. At 2 or
3 d after treatment, cells were analyzed for viability by the 3-(4,5-dimethylthiazol-
2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
(MTS) assay (Promega). For coculture experiments, MSCs-stTRAIL or U87-
stTRAIL cells were plated in the Transwell inserts containing 0.4-Am pores
(Corning Costar) withincr easing cell concentrations (0.2 104, 0.5 104,
1 104, 2 104, and 4 104 per well) and then U-87MG cells (2 104)
were grown in the lower well of the Transwell plates. For inhibition studies,
MSCs-stTRAIL (2 104) were seeded in the upper well and U-87MG cells
(2 104) were seeded in the lower well of the Transwell plates, and then
neutralizing antihuman TRAIL antibody (R&D Systems) was added to the
lower wells as indicated in the figure. After 5 d, the viability of U-87MG cells
in the lower well was analyzed by MTS assay. All experiments were
conducted in triplicate. To detect apoptotic activity, MSCs-stTRAIL and
U-87MG cells (2 104 each) were premixed and cultured in four-well
chamber slides (Nalge Nunc International) for 48 h and then stained using
a terminal deoxyribonucleotidyl transferase–mediated dUTP nick end
labeling (TUNEL) assay kit (Roche) developed with Cy3-conjugated
streptavidin ( Jackson ImmunoResearchL aboratories).