Differentiation of hNPCs. Differentiation of hNPCs in normoxic and hypoxic conditions. Cell cycle analysis by FACS did not reveal differences in the distribution of the proportion of cells in each cell cycle phase differentiated in 20% oxygen (A, n = 2-4 in duplicates) or 3% oxygen (B, n = 3-4 in duplicates). By measuring βIII-tub+ cells up to 4 days of differentiation and EPO-treatment, the percentage of neurons was evaluated in different culture conditions. Proliferation of cells at 20% and differentiation at 20% resulted at day 3 in an increase of βIII-tub+ cells compared to day one (C, n = 3-9). Cells proliferated at 3% oxygen and then differentiated at 3% and 20% oxygen showed a higher number of neurons at day 3 and day 4 (D, n = 3-5), but EPO treatment did not alter the expression levels in any of the tested conditions. (E) shows the comparison of the different control conditions, in which significant changes in the βIII-tubulin expression pattern occur with different oxygen levels (n = 3-9). Mean ± SEM, **p < 0.01). Oxygen levels during proliferation and differentiation are indicated in percentage e.g. 20% - 3% indicates 20% oxygen during proliferation and 3% oxygen during differentiation.