Mesenchymal stromal cell-associated migrasomes: a new source of chemoattractant for cells of hematopoietic origin

Abstract

Background: Multipotent mesenchymal stromal cells (MSCs) are precursors of various cell types. Through soluble factors, direct cell-cell interactions and other intercellular communication mechanisms such as extracellular vesicles and tunneling nanotubes, MSCs support tissue homeostasis. In the bone marrow microenvironment, they promote hematopoiesis. The interaction between MSCs and cancer cells enhances the cancer and metastatic potential. Here, we have demonstrated that plastic-adherent MSCs isolated from human bone marrow generate migrasomes, a newly discovered organelle playing a role in intercellular communication.

Results: Migrasomes are forming a network with retraction fibers behind the migrating MSCs or surrounding them after membrane retraction. The MSC markers, CD44, CD73, CD90, CD105 and CD166 are present on the migrasome network, the latter being specific to migrasomes. Some migrasomes harbor the late endosomal GTPase Rab7 and exosomal marker CD63 indicating the presence of multivesicular bodies. Stromal cell-derived factor 1 (SDF-1) was detected in migrasomes, suggesting that they play a chemoattractant role. Co-cultures with KG-1a leukemic cells or primary CD34⁺ hematopoietic progenitors revealed that MSC-associated migrasomes attracted them, a process intercepted by the addition of AMD3100, a specific CXCR4 receptor inhibitor, or recombinant SDF-1. An antibody directed against CD166 reduced the association of hematopoietic cells and MSC-associated migrasomes. In contrast to primary CD34⁺ progenitors, leukemic cells can take up migrasomes.

Conclusion: Overall, we described a novel mechanism used by MSCs to communicate with cells of hematopoietic origin and further studies are needed to decipher all biological aspects of migrasomes in the healthy and transformed bone marrow microenvironment. Video Abstract.

Keywords: Cellular adhesion; Extracellular vesicle; Hematopoietic stem cell; Intercellular signaling; Mesenchymal stromal cell; Migrasome; Motility.

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‴)

Fig. 2

Impact of matrix substrates on migrasome formation. A, B Primary human MSCs were cultured on different substrates at different concentrations as indicated or on uncoated glass surfaces for 24 h before being processed for CLSM. PFA-fixed cells were stained with fluorophore-conjugated WGA. Cells producing only retraction fibers and those with migrasomes were quantified (A, > 150 cells per replicate, n ≥ 3). The number of migrasomes per cell were quantified (B, > 50 cells per replicate, n ≥ 3). The mean and S.D. are shown in bar plots and symbols show the values of a given experiment (A), while boxes in plots represent data from 25–75th percentiles and middle line showing the median where each dot represents a cell (B). Data were compared using either Chi-Square test with Yates’ correction (A) or Mann–Whitney U test (B). N.s., not significant, *p < 0.05, ***p < 0.001

Fig. 3

F-actin is associated with both retraction fibers and migrasomes and tubulin is restricted to migrasomes. A–E Primary human MSCs were cultured on fibronectin-coated glass coverslips for 24 h before being processed for CLSM (A–D) and total internal reflection fluorescence (TIRF) microscopy (E). PFA-fixed cells were saponin-permeabilized prior immunolabeling with anti-α-tubulin antibodies and staining by SiR-Actin (A–C, E) or phalloidin (D) and fluorophore-conjugated WGA, which label F-actin and cellular membrane, respectively. The distribution of F-actin and tubulin in retraction fibers and migrasomes shown in A is highlighted in the insets (a′, a″). A 3D render of a single migrasome highlights the presence of actin and tubulin therein (B). The distribution of cytoskeleton constituents in early-stage and fully formed migrasomes (C, from left to right panels, respectively, see also Additional file 1: Fig. S1) and in cell-free, detached migrasome (D) is shown. The movement of SiR-Actin-stained actin filaments in retraction fibers of living cells is recorded by TIRF video microscopy at a layer height of 250 nm (E). White and yellow arrowheads point the movement of actin filaments within the time frame presented. Elapsed time in minutes is shown on the top-right corner. The images presented in B and E are excerpted from the Additional file 4: Video S3 and Additional file 5: Video S4, respectively. Scale bars, 10 μm (A, E); 5 μm (a′, a″); 1 μm (C, D)

Fig. 4

MSC-derived migrasomes exhibit a distinctive cell surface CD marker profile and carry intracellular vesicles. A–I Primary human MSCs were cultured on fibronectin-coated glass coverslips for 24 h before being processed for CLSM. PFA-fixed cells were permeabilized with saponin or non-permeabilized (NP) before immunolabeling for a given CD marker (A–C, E, G), specific tetraspanin proteins (TSPAN 2 and 4, F), Rab7 or α-tubulin (I) as indicated followed with the appropriate fluorophore-conjugated secondary antibody and membrane staining with fluorophore-conjugated WGA. CD166 immunolabeling shows its presence in mid- and fully formed migrasomes (B, C, arrowheads), whereas it is absent or very weakly expressed in early stage migrasomes (B). Quantification of CD166 signal in migrasomes and its comparison to cellular protein values show elevated levels of CD166 protein in migrasomes (D). The box-and-whisker plot shows data from 25 to 75th percentiles and 95% within the whiskers. Horizontal line represents the median, while each dot denotes a single migrasome. 3D render of a single migrasome presented in G highlights the presence of an intracellular pool of CD63 (H, arrowhead). J–L Transiently transfected Rab7-GFP MSCs were immunolabeled for CD63 and stained with fluorophore-conjugated WGA (white in J). A 3D render of the migrasome presented in J highlights the presence of cytoplasmic CD63 and late endosomal marker Rab7 (L, arrowhead). Cell-free, detached migrasomes are displayed (K). The images presented in H and L are excerpted from the Additional file 6: Video S5 and Additional file 7: Video S6, respectively. Scale bars, 5 μm (A–C, E–G, I–K); 2 μm (L); 1 μm (A, CD166, lower panel, H)

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′)