Figure 3. MSC transmigration kinetics and absence of lateral migration.

(A) MSC were incubated on hLMVEC for 30, 60 or 120 min, then fixed, stained and imaged as in Fig. 2. As in Fig. 1B, MSC were counted and classified according to their positions relative to endothelium: Apical, Spanning or Basal. Values represent mean ± s.e.m.. Asterisks indicate a statistically significant difference (p<0.05) as assessed by a one-way ANOVA test with a Tukey post-hoc test, n=3.

(B) MSC were subjected to live cell DIC (left panels) and fluorescence (middle panels) imaging during interaction with activated memDsRed-transfected hLMVEC (red). Still frames from the video at 0, 15, 30 and 45 min are shown. Numbers identify 6 separate MSC. Blue and yellow arrows indicate 2 MSC (#1 and #2) in the active process of transmigration as indicated in part by the expanding transmigration passageways in the endothelium; White dashed lines (middle panels, bottom row) indicate intercellular junctions between two ECs (‘EC1’ and ‘EC2’), blue dashed line indicates a paracellular gap for migration of MSC #1, yellow dashed line indicates a transcellular pore for transmigration of MSC #2. Pink and green dashed lines (left frames; MSC #3 and #5) represent the location of 2 different apical MSC in the preceding panel (i.e., lines at 15 min panel show the MSC positions in 0 min panel, etc.) and highlight a lack of significant lateral migration. Orange arrow, MSC #6 (15 min time point) indicates the protrusion of an MSC bleb against the endothelial surface. See also Video 2.

Scale bars represent 20 μm.