GSC-derived exosomes are internalized by monocytes and stimulate proliferation of CD14 negatively-sorted PBMCs.

<p>Fluorescent exosomes (A) were incubated with PBMCs for 5 hours and the uptake by CD14+ monocytes, CD4+ or CD8+ T cells was measured by flow cytometric analysis in the bulk population. In representative cytometry histograms, the isotype control is in black and in grey are PBMCs cells incubated with labelled-exosomes and gated on CD14+ monocytes (left, top), on CD4+ (left, bottom) or on CD8+ T cells (right, bottom). (B) Gating and sorting strategies of PBMCs and CD14-depleted PBMC. (Top, left) physical parameters, i.e. forward scatter (FSC) and side scatter (SSC), were used to select PBMCs (gate R1). Monocytes were recognized by evaluating, in PBMCs, the expression of CD14 (gate R2, top, right panel). A PE-isotype matched antibody was used to define R2 (top, central panel). PBMCs-depleted cells were identified as cells included in R1 but not in R2. (C) Representative dot-plots showing the reanalysis of the FACS-sorted PBMCs (left panel) and CD14-depleted PBMCs (right panel). As expected, CD14-positive cells were present in the sorted PBMCs- but not in the CD14-depleted- samples. (D) Proliferation of both unfractioned PBMCs and PBMCs depleted of the CD14+ population (CD14-) was measured, after CFSE labelling assay, by flow cytometry. Cells were pre-incubated without (white column, CTRL) or with (black column, GSC-EXO) GSC-derived exosomes. Columns, mean (n = 6); bars, SD; *, significantly different from the control; P<0.05. Representative cytometry CFSE histograms of PBMCs (C-E) and CD14- cells (D-F) are shown with the percentage of proliferative cells indicated.