Feeder-free SMINS-TTF-3 cells (FF SMINS3). TTF were seeded on PDL coated dishes in stem cell medium containing Bix01294, RG108, and PD0325901 for two weeks and then transferred into NSC medium in petri dishes for two weeks. (a) The FF SMINS3 cells were stained by neural stem cell markers Sox2 and Nestin. (b–f) The FF SMINS3 cells differentiated to astrocytes (GFAP), neurons (Tuj1, Map2, and NeuN), and oligodendrocytes (Olig2 and O4) in specific differentiation medium. DAPI was used for nuclei counterstaining (blue). Scale bar: 100 μm (a) and 50 μm (b–f).