Fig. 3

NESC-spheres cultured in a stirred suspension bioreactor. a Schematic representation of NESC-spheres cultured in the CHbFSB medium using a stirred suspension bioreactor. b Phase-contrast of NESC-spheres from day 0 to 3 in a suspension stirred bioreactor. c The diameter dynamics of the NESC-spheres in the suspension stirred bioreactor at the days 1, 2 and 3. Data are represented as mean ± SD (n = 3 independent experiments). d Viability of different passaged NESC-spheres (n = 3). e The NESC-spheres maintained normal karyotype after expansion of 23 passages. f Immunostainings show that NESC-spheres in the stirred suspension bioreactor express NESC markers, SOX1, NESTIN, SOX2, and PAX6, and the proliferation-associated marker Ki-67. In contrast, NESC-spheres are negative for GFAP and expressed TUJ1 in a small number of cells. g Quantification of PAX6, SOX1, and SOX2 positive cells in passage 4 NESC-spheres. Data are represented as mean ± SD (n = 3 independent experiments). No significant difference (p > 0.05) was found. Scale bars: b 200 μm; f 50 μm