Eat 2

For cell surface staining, cells were stained with fluorochrome-conjugated or biotinylated Abs against CD11b (M1/70, BioLegend), F4/80 (BM8, BioLegend), CD169 (3D6.112, BioLegend), SIGN-R1 (eBio22D1, eBiosciences), CD53 (OX-79, BioLegend), CD9 (MZ3, BioLegend), CD37 (Duno85, BioLegend), Tim1 (RMT1–4, BioLegend), Tim 2 (F37–2C4, BioLegend), Tim 3 (RMT3.23, BioLegend), Tim 4 (RMT4–54, BioLegend), CD63 (NVG-2, BioLegend), CD81 (Eat-2, BioLegend), and ADAM10 (139712, R&D Systems). Biotin labeled antibodies were visualized with fluorochrome-conjugated streptavidin (Thermo). LIVE/DEAD^® Fixable Aqua Stain (Thermo) was used to exclude dead cells. For phosphorylated protein expression assays, cells were analyzed with Abs against phospho-p38 (Thr¹⁸⁰/Tyr¹⁸²) (28B10, Cell Signaling) and total p38 (#9212, Cell Signaling), and detected using mouse or rabbit Alexa Flour 647 respectively. Data acquisitions were done on a FACSCanto II (BD) flow cytometer and analyzed with FlowJo software version 9 or 10 (Treestar).

Extracellular vesicles were prepared by ultracentrifugation as described above and washed once with PBS. EVs were then pulled down by incubating with anti CD81-Biotin (Eat-2, Biolegend) coated streptavidin beads overnight rotating at 4°C (Exosome-Streptavidin Isolation/Detection reagent, Invitrogen). Beads were then collected using a magnet and washed 3 times with PBS supplemented with 0.1% BSA. For fluorescent labeling, pulled down EV/Bead complexes were stained using anti CD9-AF647 (MZ3-Biolegend) and analyzed using BDFACS ARIA II. For PKH-26 (Sigma-Aldrich) labeling, 200ug of ultracentrifugation enriched EVs were pulled down using anti-CD81 coated Exobeads as described above. Captured EVs were labeled in 200ul volume for five minutes. Labeling was stopped using an equal volume of PBS with 1% BSA and samples were washed three times according to manufacturer’s instructions. The equivalent of 100ug starting material of Exobead captured EVs labeled with PKH-26 were added to 50K sorted GMPs in StemSpan supplemented with 1% Penicillin/Streptomycin and L-Glutamine without cytokines. Cells were analyzed 12 hours later for protein translation and cellular proliferation.