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sc-12-0231_sm_SupplFigure1.tif849KFigure S1. DMSO eliminates undifferentiated cells in cultures of hESC-OPs. (A) Undifferentiated cells (OCT4-positive cells) were detected in cultures with differentiation medium without DMSO. Scale bars = 30μm. (B) Western blot of OCT4 in hESC-OPs after supplementing the medium with 1% DMSO. After seven days of culture, OCT4 expression became negligible.
sc-12-0231_sm_SupplFigure2.tif870KFigure S2. Oligodendrocyte differentiation of hESC-OPs in vitro. Differentiated hESCOPs were positive for CNPase (A), the oligodendrocyte marker O4 (B), and galactocerebroside- (C). Scale bars = 40μm.
sc-12-0231_sm_SupplFigure3.tif1978KFigure S3. Representative MRI of transplanted hESC-derived OPs in EAE mice. Serial ex vivo MRI of labeled hESC-derived OPs following ICV delivery was performed at day 14 post-EAE induction. On day 10 PT, hypointense signals were observed mainly in the ventricles (A, D, H). A few hypointesities were detected in the white matter tract (D). A similar distribution pattern was observed in images taken on day 20 (B, F, I), and 30 PT (C, G, J). MRI parameters: FLASH; TR= 150 ms; TE=8 ms; resolution=65x65x65 μm; average=2.
sc-12-0231_sm_SupplFigure4.tif1420KFigure S4. Transplanted hESC-OPs migrate to spinal cords within the CSF. PCR analysis shows that human-specific genes (SRY and TPOX) were expressed in the cerebrum (lane 3), cerebellum (lane 4), cervical (lane 5), thoracic (lane 6), and lumbar (lane 7) spinal cord at 5 days PT. hESC-OPs (lane 1), and normal mouse brain without human cells (lane 2) were used as positive and negative controls, respectively.
sc-12-0231_sm_SupplFigure5.pdf223KFigure S5. Transplanted hESC-OPs increase Foxp3 activation in the spinal cord and spleen. (A) Foxp3 immunostaining in the spinal cord. In EAE control animals, Foxp3- positive cells were detected in the subarachnoid space (arrowheads) and white matter (arrow) after day 15 PT (day 22 post-EAE induction). In the group that received live hESC-OPs, Foxp3-positive cells (arrowheads) were seen in the subarachnoid space at days 5, 15, and 25 PT. Scale bars = 50 μm. (B) Semi-quantitative analysis of Foxp3- positive cells for EAE control and animals that received live hESC-OPs at day 25 PT. Data represent the mean of the number of Foxp3-positive cells ± SEM. *p <0.05 vs EAE control. (C) Representative photographs of Foxp3 and β-actin in the spleen at day 25 PT. (D) Semi-quantitative analysis of Foxp3 activity in the spleen, normalized to the intensity of β-actin expression in the same immunoblot. Data represent the mean of optical density ± SEM. *p <0.05 vs. EAE control.
sc-12-0231_sm_SupplFigure6.tif1169KFigure S6. Transplanted hESC-OPs increase GFAP activity in the glia limitans. GFAP immunoreactivity was detected in the spinal cords of normal control mice, and its reactivity was increased in the spinal cords of EAE animals. Enhanced GFAP activity was detected in the spinal cords of animals that received cells at day 15 PT. Merged images represent GFAP (red) and Hoechst (blue) staining. Arrows indicate the glia limitans. Scale bars = 100 μm.

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