Neuronal EVs promote neural induction and cell proliferation of mESCs. (A) Gene expression analysis of Nestin and Six3 in mESCs treated without (control) or with indicated EVs. EV number was quantified by Nanosight 2000. Data plotted were from three independent experiments, each with triplicate qPCR reactions; error bars represent SD from independent samples. The values represent the mean ± SD (*, P < 0.05). (B) Gene expression analysis of Nestin and Six3 in mESCs treated for 4 d without (control) or with different doses of N6-EV. Data plotted were from three independent experiments, each with triplicate qPCR reactions; error bars represent SD from independent samples. The values represent the mean ± SD, from three independent experiments (*, P < 0.05; **, P < 0.01). (C) Gene expression analysis of Sox1, Pax6, and Tuj1 in mESCs treated for 4 d with 2× 10⁹ PC12-EV, N6-EV, or N6-EV together with NGF neutralizing antibody (500 ng/ml). The values represent the mean ± SD, from three independent experiments (*, P < 0.05; NS, P > 0.05). Error bars represent SD from independent samples. (D) Immunostaining of Nestin (green, Alexa Fluor 488) and Pax6 (red, Alexa Fluor 568) in mESCs as described in B. Scale bar, 25 µm. (E) Quantitative analysis of the percentage of cells containing indicated markers compared with DAPI-stained cells. The values represent the mean ± SD, from three independent experiments (*, P < 0.05). Error bars represent SD from independent samples. (F) Gene expression analysis of Sox1, Pax6, and Nestin in mESCs treated for 4 d with 2 × 10⁹ EVs from undifferentiated pluripotent ESCs (ES-D0), EBs of ES differentiated for 8 d in KSR medium (ES-D8), and EBs trypsinized in N2 medium for an additional 4 d (ES-D12). The values represent the mean ± SD, from two independent experiments (*, P < 0.05; NS, P > 0.05). Error bars represent SD from independent samples. (G) The cellular morphology of mESCs treated with PC12-Exo or different doses of N6-EV in N2B27 medium for 4 d. Scale bars, 200 µm. (H) Quantitative analysis of mESC number treated with or without N6-EV. Data plotted were from two independent experiments. The values represent the mean ± SD (*, P < 0.05). Error bars represent SD from independent samples. (I) Proliferation analysis of mESCs with BrdU staining after EV treatment. The values represent the mean ± SD, from two independent experiments (*, P < 0.05). Error bars represent SD from independent samples.