Fig. 2

Long-term expanded NESC-spheres maintained NESC characteristics. a Immunofluorescence staining of cryosections showed that NESC-spheres from passage 5 at day 3 express NESC markers, such as SOX1, NESTIN, SOX2, and PAX6, and the proliferation-associated marker Ki-67. In contrast, NESC-spheres are negative for GFAP and express TUJ1 in a small number of cells. b–d Quantification of the number of SOX1 (b)-, SOX2 (c)-, and PAX6 (d)-positive cells in the passage 5, 15, 25 NESC-spheres, respectively. e Quantification of differentiated TUJ1-positive neurons in the passaged 5, 15, 25 NESC-spheres, respectively. f Schematic representation of single NESCs from spheres self-organization into neural tube (NT)-like structures in the colony assays. g Single NESCs in one well of the 96-well plate at day 0. h Representative NT-like structures of NESCs at day12. i–m The polarized NTs express NESC markers, such as NESTIN, N-CADHERIN, SOX1, SOX2, PAX6, and ZO-1. b–e Data are represented as mean ± SD (n = 3 independent experiments). *p < 0.05, ***p < 0.001, ****p < 0.0001 by Student’s t test. Scale bars: g, h 100 μm; others, 50 μm