Fig 1: (a) Schematic representation of the differentiation protocol. Exposure to Basal Medium supplemented with 5 mM of fresh lithium chloride for 8 days induced hiPSCs to differentiate into NCCs. Neural crest cells were then exposed to normoxic or hypoxic conditions for 48 and 72 hours to evaluate their capacity to secrete functional EPO. (b, c) Immunostaining analysis for neural crest cells specific markers (b) Sox10, HNK1 and (c) AP2? at day 8 of differentiation. (d) Bright field microscopy image of migrating cells at day 7 of differentiation. (e, f) Immunofluorescent images of EPO production in (e) normoxic and (f) hypoxic conditions. EPO is secreted in cell cytoplasm (inset). (g) EPO concentration in the supernatant of undifferentiated hiPSCs (d0, control) and NCCs under normoxic or hypoxic conditions for 48h and 72h. ***p<0.001 vs all culture conditions tested by one - way ANOVA. N, normoxia; H, hypoxia; h, hours. Digital images (b, c, e, f) were acquired using an inverted confocal laser microscope (Leica TCS SP8, Leica Biosystems) or (d) Apotome Axio Imager Z2 (Carl Zeiss). Scale bars (b, c) 10 µm, (d) 100 µm, (e, f) 50 µm. More information about NCCs’ characterization and immonofluorescence protocols can be found in the original paper.