Following MACS sorting, NCSCs were replated onto 48-well plates in E6-CSFD medium + 10 μM Y27632. Cells were switched to mural cell differentiation medium the next day, expanded for 6 days, and stained for NG2/PDGFRβ expression. Cells were expanded on uncoated plates in E6 medium, E6 medium supplemented with TGFβ1 (2 ng/ml) + PDGF-BB (20 ng/ml), or E6 medium supplemented with 10% FBS. Cells were also expanded in E6 supplemented with 10% FBS on gelatin-coated plates prepared by coating plates for at least 1 hour at 37°C with a 0.1% gelatin A solution dissolved in water.

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