However, we noticed different cellular morphologies and densities between AD-NPC and N-NPC cultures immediately after the initiation of neuronal differentiation (Figure 2a). Many cells of AD-NPCs exhibited the typical neuron morphology with long neurite outgrowth, whereas the majority of cells of N-NPCs maintained a typical NPC morphology (Figure 2a). To assess the percentages of NPCs and neurons in the differentiation culture, we carried out immunofluorescence staining using antibodies against Nestin and MAP2 at day 28 of differentiation. Significantly lower percentages of Nestin+ NPCs but higher percentages of MAP2+ neurons were found in AD-NPCs, compared to those in N-NPCs (Figure 2b and 2c). Similar results were obtained from differentiating cells at other days examined (Figure 2d and Figure 1l). Moreover, the number of Nestin+ cells in the AD-NPCs culture was significantly less than that in N-NPCs during differentiation from day 28 to 36 (Figure 2d). In addition, we detected higher percentages of neurons and lower percentages of NPCs in AD-NPCs generated through a widely used SMAD inhibition neural differentiation protocol [26, 27], suggesting that the phenotype of premature neuronal differentiation observed during differentiation of AD-NPCs is not dependent on the NPCs generation strategy (Figure 1m).
