Fig. 5

MSC-derived migrasomes can act as a chemoattractant organelle. A–K Primary human MSCs were cultured on fibronectin-coated glass coverslips under sub-confluent state for 24 h prior to, either, imaging (A–C), or addition of KG-1a (k) cells (D–I, K) or primary CD34⁺ HSPCs (J) for 4 (E–G, I, J) or 12 (D–F, H, K) h co-culture as indicated. Afterward, they were processed for CLSM (A, B) or live-cell microscopy (C–K). CD34⁺ HSPCs were used 24 (red) or 48 (black) h after their immunoisolation (J). SDF-1 was detected by immunolabeling (A) or upon transient transfection with SDF-1-GFP (B), followed by staining with fluorophore-conjugated WGA. SDF-1 can be observed in migrasomes (yellow arrowhead, a′, b′) or retraction fibers (a″, asterisk). Note that not all migrasomes contained SDF-1 (white arrows). Time-lapse video of an MSC-associated migrasome network shows the migrasomes detaching from substrate (C, c′, red asterisks) or releasing their contents into medium as suggested by phase-shift (c′, black → yellow asterisks). The boxed regions in C indicate the areas enlarged in the insets. The elapsed time in minutes is shown on the top right corner. All frames are excerpted from Additional file 9: Video S8. The areas covered by MSCs (MSC), the migrasome network (MN), and free surface (FS) are shown (D, E), while the distribution of KG-1a cells or CD34⁺ HSPCs on these regions was quantified after 4 (E, F, J) or 12 (D–F) h of co-culture with MSCs. Data are presented as the fold change of the density of KG-1a cells or CD34⁺ HSPCs per mm² in regard to FS; dashed red line indicates same density with FS, thereby no preferential localization (F–J). KG-1a cells were co-cultured with MSCs in the presence of either 1 or 10 µM AMD3100 or 100 ng/mL recombinant SDF-1 for 4 h and their distribution were quantified as in panel F (G). Alternatively, MSCs were pre-treated with anti-CD166 antibody, or IgG control, for 2 h prior to addition of KG-1a (I) cells or CD34⁺ HSPCs (J). Error bars shows the S.D., while symbols show the individual experiments (F, G, I) or donors (J). More than 200 cells were analyzed for each experiment (n = 3). Mean of experiments were compared against control using unpaired T test (G, I) or paired T test (J, note that only 48-h values were used for statistical analysis). Migrating KG-1a cells can either move towards a migrasome network and be captured by retraction fibers (H) or uptake the migrasomes during their movement (K, arrowheads). Green and white dashed outlines show the position of migrasome network (H) and the cell in the previous frame (H, K), respectively. White and yellow arrowheads show the migrasomes that are taken by KG-1a cell (K). All frames are excerpted from Additional file 10: Video S9 and Additional file 11: Video S10, respectively. N.s., not significant, *p < 0.05, **p < 0.01, ***p < 0.001. Scale bars, 10 μm (C, D, H, K); 5 μm (A, B, a″, c′); 1 μm (a′, b′)