Figure 6
Impact of EL4 and EL4-v10 on bone marrow stroma-promoted HSC quiescence. (A,B) CFSE-labeled HSC (1×106) were cocultured with EL4 or EL4-v10 exosomes (10 μg/ml) on HA, OPN or BM-Str-coated plates for 4 h. After removing non-adherent cells, cells were lysed and fluorescence was determined photometrically. (A) Percent HSC (mean ± SD, triplicates) adhering to HA, OPN or BM-Str in dependence on EL4/EL4-v10 exosomes; significant differences (p <0.05) by EL4 or EL4-v10: s; (B) percent HSC (mean ± SD, triplicates) adhering to HA, OPN or BM-Str in dependence on IM7 or K926; significant (p <0.05) antibody inhibition: *; (C) HSC (1×106) were seeded on BSA, HA or BM-Str coated 24-well plates and starved for 24 h. Thereafter HSC were cultured for 2 h in medium containing 10% FCS; where indicated cultures contained EL4/EL4-v10 exosomes (10 μg/ml) or IM7 or K926 (10 μg/ml). HSC were collected and treated as described in Figure 4D; PI staining was evaluated by flow cytometry; the mean percent (triplicates) of cells in G0, G1/S and G2/M and representative examples are shown; significant impact (p <0.05) of BM-Str: *; significant differences (p <0.05) by EL4 or EL4-v10: s, significant differences (p <0.05) by antibody: *; (D-F) CFSE-labeled HSC (1×106) were cultured on HA or BM-Str coated 24-well plates for 24 h; cultures contained EL4 or EL4-v10 exosomes (10 μg/ml) or IM7 or K926 (10 μg/ml). HSC were collected and proliferation was evaluated by CFSE dilution (flow-cytometry), the mean% (triplicates) of HSC that had not divided or divided 1-times or 2-times and representative examples are shown; significant differences (p <0.05) by BM-Str as compared to HA: *, significant differences (p <0.05) by EL4 / EL4-v10 exosomes: s, significant differences (p <0.05) by antibody: *. Experiments were repeated 3-times. EL4, more pronounced than EL4-v10 interfere with HSC – BM-Str adhesion and HSC quiescence, which is also disturbed by IM7.