Fig 2: Human CD63-GFP expression and localization during differentiation of eNSCs obtained from the telencephalon of Tg rats at E14. (A) Schematic representation of the protocol for eNSC proliferation and differentiation in vitro. (B) GFP-positive neurospheres from Tg rats. Scale bar: 50 µm. (C) Protein expression profiles during differentiation of eNSCs. Markers for neurons (TUJ1), astrocytes (GFAP), oligodendrocytes (CNPase), neural stem cells (SOX2) and presynaptic (syntaxin 1) and postsynaptic (GluR1) proteins, rat CD63 and human CD63-GFP were examined in Wt and Tg rat cells. The experiment was performed once. (D) GFP expression in the cultured Tg rat cells at day 0 (left) and 5 days after differentiation (right). Immunostaining indicated the co-localization of GFP with human CD63-positive and with rat CD63-positive signals around nuclei (blue) in the nestin- and SOX2-positive undifferentiated cells at day 0. At day 5, no GFP signal was seen in the MAP2-positive, CNPase-positive and GFAP-positive differentiated cells (arrows). All the experiments except (C) were repeated at least two times using different cultures. Scale bars: 20 µm.

Fig 3: In vitro transfer of neurosphere-derived EVs to astrocytes in the co-culture system. (A) A schematic illustration of the co-culture protocol. Wt astrocytes as recipient cells were cultured on pre-coated coverglass on the bottom of wells, and then they were cultured for 4 days with Tg or Wt neurospheres put into the culture insert with 1.0 µm pores. (B) EVs derived from Tg neurospheres in the Wt astrocytes were detected by GFP (green) and immunoreactivity of human CD63 (red). Astrocytes were stained with an antibody against GFAP (cyan). Nuclei were visualized by DAPI (blue). All the experiments were duplicated in different plates using sister cultures. Scale bars: 50 µm.

Fig 4: Identification and characterization of the EVs isolated from the serum of Wt and two Tg rats [Wistar-esTgN(Sox2/CD63-GFP)3NCCRI and Wistar-esTgN(CAG/CD63-GFP)3NCCRI]. (A) Size distribution of the EVs collected by ultracentrifugation was determined using the NanoSight system. (B) Western blot analysis of the EVs for flotillin-1, rat CD63, human CD63 and copGFP (n=2).

Fig 5: Generation of Tg rats by transfection of Sox2/human CD63-GFP gene into rESCs. (A) The localization of Sox2 promoter including two BglII sites (left) and structure of the transgene construction (right). (B) Photomicrograph of No. 6 rESC line transfected with the Sox2/human CD63-GFP gene. The rESCs expressed GFP ubiquitously. Scale bars: 300 µm. (C) E14 rats obtained by mating a Tg [Wistar-esTgN(Sox2/CD63-GFP)3NCCRI] male rat with a Wt female rat. The fetus not expressing GFP is a non-Tg littermate (i). The Tg fetus showed a high level of GFP fluorescence in the spinal cord (ii, arrowhead) and telencephalon (ii, arrow, iii and iv). Scale bars: 5 mm. (D) Western blot analysis for SOX2, endogenous rat CD63, exogenous human CD63 and copGFP during rat brain development. The lysates were collected from the telencephalon at E14 and the cerebral cortex at E19, P4 and P10 (n=3). ß-actin was used as a loading control. Error bars represent the s.d. (*P<0.001 versus E14, one-way ANOVA with Bonferroni's post hoc test).