Fig. 1
Primary human mesenchymal stromal cells produce migrasome networks. A–J Primary human MSCs were cultured on fibronectin-coated glass coverslips for 24–72 h before being processed for CLSM (A–E), SEM (F–H), or live-cell phase-contrast video microscopy (I, J). After 24 h in culture, PFA-fixed cells were stained with fluorophore-conjugated WGA (A–C). Early-stage and fully formed migrasomes that develop along the retraction fibers left behind a migrating MSC are indicated by black and white arrowheads, respectively. Migrasomes are also found at the tips of retraction fibers (B, asterisk). Over time, the retraction fibers breakdown, leaving the migrasomes free (B, C, arrow and inset). The number of cells with migrasomes and the number of migrasomes per cell (D) were quantified after 24, 48, and 72 h of culture. Cell-free, detached migrasomes per mm2 were quantified over time in cultured cells (E). The mean and S.D. of all data are shown in bar plots and symbols show the values of a given experiment (D, left, E), while box-and-whisker plots show data from 25 to 75th percentiles and 95% within the whiskers. Horizontal line represents the median, where each dot represents a cell (D, right) (> 150 cells per replicate, n = 3). 48-h cultured MSCs were processed for SEM (F–H). Note that migrasomes often develop on the branching points of the retraction fibers or at their tips (F, G, inset g′, g”, H, upper panel) and are released upon degradation of the fiber network (H, lower panel). The formation of migrasomes is revealed by time-lapse video imaging of MSCs (I, J). After 24 h in culture, the dynamics of MSCs and the growth of migrasomes were recorded for a period of 12 h. Elapsed time in minutes is shown on the top-right corner. Dashed white and black arrows indicate the direction of MSC membrane retraction or the cell migration, respectively, while dashed outline shows the extent of the migrasome network. The migrasome maturation is highlighted over time in panels J (insets) and J (insets j′, j″, j‴) and their release is indicated (double arrowhead). The images presented in I and J are excerpted from the Additional file 2: Video S1 and Additional file 3: Video S2, respectively. K Migrasome biogenesis by MSCs is triggered by two distinct cellular mechanisms: cell migration and membrane retraction. In both cases, migrasomes grow on the retraction fibers and are released upon degradation of the fiber network. Data were compared using either Mann–Whitney U test (D, E). ***p < 0.001. Scale bars, 10 μm (A–C, I, J); 5 μm (H, upper panel); 1 μm (F, G, H, lower panel, j′–j‴)