Fig. 3

Validation and characterization of AiNPCs. a-d Bright-field of AiNPCs showed a typical NPC morphology in both suspension and adherent cultures, compared to control NPCs. e-h AiNPCs expressed NPC markers Nestin, Sox2, and Pax6. i Nuclear-cytoplasmic ratios of AiNPCs, control astrocytes, and control NPCs were assessed by quantifying the size of nucleus and cytoplasm in Image-Pro Plus. More than 100 cells per groups were randomly chosen for each measurement. **P < 0.01 by two-tailed t test (n = 3). j Proliferation of AiNPCs and control NPCs were determined by counting the total cell number during each passage. Mdt, mean doubling time. Sd, standard deviation. Btw psg, between passages. k Total neurosphere number per 100 AiNPCs or control NPCs were counted to determine the neurosphere forming efficiencies. l, m RNA of AiNPCs, control astrocytes, and control NPCs were collected and the expression of astrocytic differentiation marker genes (l) and NPC marker genes (m) was analyzed by qPCR. Data were normalized to GAPDH and presented as fold change compared with control astrocytes. n-v Control NPCs and AiNPCs were placed in neuronal (n, q, t u, v), astrocyte (o, r), and oligodendrocyte (p, s) differentiation media. Cultures were fixed and stained with Tuj1 (n, q), GFAP (o, r), O4 (p, s), Tau (t), and MAP2/synaptophysin (u, v). (w) The Nestin promoter regulatory region DNA methylation patterns of AiNPCs, control astrocytes, and control NPCs were analyzed using a pyrosequencing method. *Human lymphocyte genomic DNA was used as a negative control for pyrosequencing. **Sss1 methyltranferase treated human lymphocyte genomic DNA was used for positive control. Scale bars represent 10 μm (a-h, n-v). Error bars denote s.d. from triplicate measurements (i, k, l, m)