Figure 6. Fibronectin Associated with EVs from ESCs Is Important for Promoting Stemness.

(A) ESCs cultured in 2i+LIF or N2B27 medium, as well as the MVs and exosomes isolated from these cells, were immunoblotted for fibronectin, the cytosolic protein FAK, and the general EV marker Flotillin-2.*

(B) MVs and exosome (Exo) preparations isolated from ESCs using the procedure described in Figure S1G were further resolved by sucrose density gradient ultracentrifugation. The intermediate-density fraction collected from each preparation was lysed and immunoblotted for fibronectin and the general EV marker Flotillin-2.

(C) ESCs maintained in N2B27 medium supplemented without (untreated) or with MVs and exosomes isolated from ESCs cultured in either 2i+LIF medium (2i+LIF MVs+Exo) or N2B27 medium (N2B27 MVs+Exo) were immunoblotted for Oct3/4, Nanog, H3K27me3, and vinculin as the loading control. ESCs grown in 2i+LIF medium were used as the positive control (first lane).

(D) ESCs cultured in N2B27 medium supplemented with MVs and exosomes from pluripotent ESCs and treated without (control) or with 25 μg/mL of the RGD peptide were immunoblotted for phosphorylated FAK (p-FAK) and total FAK.*

(E) ESCs cultured in N2B27 medium supplemented with MVs and/or exosomes from pluripotent ESCs and treated without (control) or with 25 μg/mL of the RGD peptide were immunoblotted for Oct3/4, Nanog, and vinculin as the loading control.*

(F) The amount of MVs and exosomes from ESCs that was used to promote stemness and the increasing amounts of purified fibronectin that were immunoblotted for fibronectin.

(G) ESCs cultured in N2B27 medium supplemented without (untreated) or with 320 ng of purified fibronectin (FN) were immunoblotted for Oct3/4, Nanog, and vinculin as the loading control. ESCs grown in 2i+LIF medium were used as the positive control (first lane).*

(H) Images of sphere formation assays performed on ESCs cultured in N2B27 medium supplemented without (untreated) or with 320 ng of purified fibronectin (FN) or MVs and exosomes from pluripotent ESCs. Scale bar, 100 μm.

(I) Quantification of the assays shown in (H).*

(J) ESCs cultured in N2B27 medium supplemented without (untreated) or with vesicle-free medium (VFM), or MVs and exosomes from ESCs (MVs+Exo), were immunoblotted for Oct3/4, Nanog, and vinculin as the loading control. ESCs grown in 2i+LIF medium were used as the positive control (first lane).*

*The data shown in (A), (C)–(E), (G), (I), and (J) are presented as mean ± SD. All experiments were performed at least three independent times, and statistical significance was determined using Student’s t test; ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; and ns, not significant. See also Figures S6 and S7.